Modified ciliary neurotrophic factor, method of making and methods of use thereof

ABSTRACT

Modified ciliary neurotrophic factor, methods for production and methods of use, especially in the treatment of Huntington&#39;s disease, obesity, and gestational or adult onset diabetes.

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 08/645,107 filed May 13, 1996, which is acontinuation-in-part of Ser. No. 08/308,736 filed Sep. 14, 1994, whichis a continuation-in-part of U.S. patent application Ser. No. 07/959,284filed Oct. 9, 1992 entitled “Ciliary Neurotrophic Factors” which issuedas U.S. Pat. No. 5,349,056 on Sep. 20, 1994. Throughout thisapplication, various patent and publications are referenced. Thosepatents and publications are hereby incorporated by reference in theirentireties, into this application.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to therapeutic CNTF-relatedpolypeptides useful for the treatment of neurological or other diseasesor disorders.

[0003] Ciliary neurotrophic factor (CNTF) is a protein that is requiredfor the survival of embryonic chick ciliary ganglion neurons in vitro(Manthorpe et al., 1980, J. Neurochem. 34:69-75). The ciliary ganglionis anatomically located within the orbital cavity, lying between thelateral rectus and the sheath of the optic nerve; it receivesparasympathetic nerve fibers from the oculomotor nerve which innervatesthe ciliary muscle and sphincter pupillae.

[0004] Over the past decade, a number of biological effects have beenascribed to CNTF in addition to its ability to support the survival ofciliary ganglion neurons. CNTF is believed to induce the differentiationof bipotential glial progenitor cells in the perinatal rat optic nerveand brain (Hughes et al., 1988, Nature 335:70-73). Furthermore, it hasbeen observed to promote the survival of embryonic chick dorsal rootganglion sensory neurons (Skaper and Varon, 1986, Brain Res. 389:39-46).In addition, CNTF supports the survival and differentiation of motorneurons, hippocampal neurons and presympathetic spinal cord neurons[Sendtner, et al., 1990, Nature 345: 440-441; Ip, et al. 1991, J.Neurosci. 11:3124-3134; Blottner, et al. 1989, Neurosci. Lett.105:316-320].

[0005] It has long been known that innervation of skeletal muscle playsa critical role in the maintenance of muscle structure and function.Skeletal muscle has been shown recently to be a target of positive CNTFactions. Specifically, CNTF prevents both the denervation-inducedatrophy (decreased wet weight and myofiber cross sectional area) ofskeletal muscle and the reduced twitch and tetanic tensions ofdenervated skeletal muscle. Helgren et al., 1994, Cell 76:493-504. Inthis model, human CNTF also produces an adverse effect that ismanifested as a retardation of weight gain. This adverse effect has alsobeen observed in clinical trials with rHCNTF for the treatment of ALS.Therefore, simultaneous measurements of muscle weight and animal bodyweight following denervation could be used as a measure of efficacy andadverse reaction, respectively, in response to treatment with rHCNTF orother compounds. The ratio of the potency values obtained from thesemeasurements is defined as the therapeutic index (T.I.), expressed hereas TD₂₅/ED₅₀, so that the higher the value of T.I., the safer thecompound at a therapeutic dose.

[0006] CNTF has been cloned and synthesized in bacterial expressionsystems, as described by Masiakowski, et al., 1991, J. Neurosci.57:1003-1012 and in International Publication No. WO 91/04316, publishedon Apr. 4, 1991, which are incorporated by reference in their entiretyherein.

[0007] The receptor for CNTF (termed “CNTFRα”) has been cloned,sequenced and expressed [see Davis, et al., 1991 Science 253:59-63].CNTF and the hemopoietic factor known as leukemia inhibitory factor(LIF) act on neuronal cells via a shared signaling pathway that involvesthe IL-6 signal transducing component gp130 as well as a second,β-component (know as LIFR β); accordingly, the CNTF/CNTF receptorcomplex can initiate signal transduction in LIF responsive cells, orother cells which carry the gp130 and LIFRβ components [Ip, et al.,1992, Cell 69:1121-1132].

[0008] In addition to human CNTF, the corresponding rat (Stockli et al.,1989, Nature 342:920-923), and rabbit (Lin et al., 1989, J. Biol. Chem.265:8942-8947) genes have been cloned and found to encode a protein of200 amino acids, which share about 80% sequence identity with the humangene. Both the human and rat recombinant proteins have been expressed atexceptionally high levels (up to 70% of total protein) and purified tonear homogeneity.

[0009] Despite their structural and functional similarity, recombinanthuman and rat CNTF differ in several respects. The biological activityof recombinant rat CNTF in supporting survival and neurite outgrowthfrom embryonic chick ciliary neurons in culture is four times betterthan that of recombinant human CNTF [Masiakowski et al., 1991, J.Neurochem. 57:1003-1012]. Further, rat CNTF has a higher affinity forthe human CNTF receptor than does human CNTF.

[0010] A surprising difference in the physical properties of human andrat CNTF, which are identical in size, is their different mobility onSDS gels. This difference in behavior suggests the presence of anunusual structural feature in one of the two molecules that persistseven in the denatured state (Masiakowski et al., 1991, J. Neurochem.57:1003-1012).

[0011] Mutagenesis by genetic engineering has been used extensively inorder to elucidate the structural organization of functional domains ofrecombinant proteins. Several different approaches have been describedin the literature for carrying out deletion or substitution mutagenesis.The most successful appear to be alanine scanning mutagenesis[Cunningham and Wells 1989, Science 244: 1081-1085] and homolog-scanningmutagenesis [Cunningham et al., 1989, Science 243:1330-1336]. Theseapproaches helped identify the receptor binding domains of growthhormone and create hybrid proteins with altered binding properties totheir cognate receptors.

[0012] To better understand the physical, biochemical andpharmacological properties of rHCNTF, applicant undertook rationalmutagenesis of the human and rat CNTF genes based on the differentbiological and physical properties of their corresponding recombinantproteins (See Masiakowski, P., et al., 1991, J. Neurochem.,57:1003-1012). Applicant has found that the nature of the amino acid atposition 63 could greatly enhance the affinity of human CNTF for sCNTFRαand its biological potency in vitro (Panayotatos, N., et al., J.

[0013] Biol. Chem., 1993, 268:19000-19003 Panayotatos, N., et al.,Biochemistry, 1994, 33: 5813-5818.

[0014] As described in copending U.S. patent application Ser. No.07/570,651 filed Aug. 20, 1990, entitled “Ciliary Neurotrophic Factor”,which is incorporated by reference in its entirety herein, one of theuses of CNTF contemplated by applicants was the use of CNTF for thetreatment of Huntington's disease. Huntington's disease (HD) is anhereditary degenerative disorder of the central nervous system. Thepathology underlying HD is progressive, relentless degeneration of thebasal ganglia, structures deep inside the brain which are responsiblefor aspects of the integration of voluntary motor and cognitiveactivity. The onset of symptoms in HD is generally in adulthood, betweenthe ages of 20 and 40. The characteristic manifestations of the diseaseare chorea and other involuntary movements, dementia, and psychiatricsymptoms. Choreic movements consist of brief, involuntary, fluidmovements, predominantly affecting the distal extremities. Patientsoften tend to “cover up” these movements by blending them into voluntaryacts. HD patients also, however, display a variety of other neurologicalabnormalities including dystonia (sustained, abnormal posturing), tics(“habit spasms”), ataxia (incoordination) and dysarthria (slurredspeech). The dementia of HD is characterized as the prototypical“subcortical” dementia. Manifestations of dementia in HD includeslowness of mentation and difficulty in concentration and in sequencingtasks. Behavioral disturbances in HD patients are varied, and caninclude personality changes such as apathy and withdrawal; agitation,impulsiveness, paranoia, depression, aggressive behavior, delusions,psychosis, etc. The relentless motor, cognitive and behavioral declineresults in social and functional incapacity and, ultimately death.

[0015] HD is inherited as an autosomal dominant trait. Its prevalence inthe U.S. population is estimated to be 5 to 10 per 100,000 individuals,yielding a total prevalence of 25,000 in the US population. However, dueto the late onset of symptoms, there are a number of “at-risk”,asymptomatic individuals in the population as well. The prevalence ofasymptomatic, at-risk patients carrying the HD gene is perhaps twicethat of the symptomatic patients (W. Koroshetz and N. Wexler, personalcommunication). Thus, the total HD patient population eligible toreceive a new therapy is about 75,000.

[0016] The gene currently believed to be responsible for thepathogenesis of HD is located at the telomeric end of the short arm ofChromosome 4. This gene codes for a structurally novel protein ofunknown function, and the relationship of the gene product to thepathogenesis of HD remains uncertain at the present time.

[0017] The principal anatomical lesion in HD consists of loss of theso-called “medium spiny” neurons of the caudate nucleus and putamen(collectively known as the striatum in rodents). These neurons comprisethe projection system whereby the caudate/putamen projects to its outputnuclei elsewhere in the basal ganglia of the brain. The principalneurotransmitter utilized by the medium spiny neurons isgamma-aminobutyric acid (GABA), although many also contain neuropeptidessuch as enkephalins and substance P. It is clear, however, that in HDinterneurons which do not utilize GABA as their neurotransmitter,containing instead either acetylcholine or the neuropeptidessomatostatin or neuropeptide Y, are relatively undamaged in HD.

[0018] Pathological and neurochemical changes which mimic those seen inHD can be mimicked by infusion of glutamatergic agonist drugs into thestriatum. Infusion of quinolinic acid under appropriate conditionsproduces selective depletion of medium sized intrinsic striatal neuronswhich utilize gamma-aminobutyric acid (GABA) as their neurotransmitter,without affecting the large, cholinergic interneurons.

[0019] There have been no successful clinical trials of eithersymptomatic or neuroprotective treatments in HD. However, useful,validated rating instruments and neuroimaging techniques exist which arecapable of monitoring disease progress and patient function.

[0020] The CNTF receptor complex contains 3 proteins: a specificitydetermining α component that directly binds to CNTF, as well as 2 signaltransducing β components (LIFR β and gp130) that cannot bind CNTF ontheir own, but are required to initiate signaling in response to CNTF.The β component of the CNTFR complex is more widely distributedthroughout the body than the α component. The 3 components of the CNTFRcomplex are normally unassociated on the cell surface; CNTF induces thestepwise assembly of a complete receptor complex by first binding toCNTFR α, then engaging gp130, and finally recruiting LIFR β. When thisfinal step in receptor assembly occurs (heterodimerization of the βcomponents), intracellular signaling is initiated by activatingnon-receptor tyrosine kinases (JAK kinases) associated with theβcomponents. JAK kinases respond by phosphorylating each other and alsotyrosine residues on the receptor cytoplasmic domains, creatingphosphotyrosine docking sites for the Src homology 2 domains of STATproteins. After their phosphorylation, bound STAT proteins dissociatefrom the receptor, dimerize, and translocate to the nucleus where theybind DNA and activate transcription (reviews: Frank, D. and Greenberg,M. (1996) Perspectives on developmental neurobiology 4: 3-18; Stahl, N.and Yancopoulos, G. (1997) Growth factors and cytokines in health anddisease 2B, 777-809). Axokine is a mutant CNTF molecule with improvedphysical and chemical properties, which retains the ability to interactwith and activate the CNTF receptor. (Panayotatos, N., et al. (1993) J.Biol. Chem. 268: 19000-19003).

[0021] Leptin, the product of the ob gene, is secreted by adipocytes andfunctions as a peripheral signal to the brain to regulate food intakeand energy metabolism (Zhang, Y., et al. (1994) Nature 372: 425-431).

[0022] Interestingly, leptin receptor (OB-R), a single membrane-spanningreceptor has considerable sequence similarities to gp130 (Tartaglia, L.,et al. (1995) Cell 83: 1263-1271), and like CNTF, leptin signals throughthe JAK/STAT pathway (Baumann, H., et al. (1996) Proc. Natl. Acad. Sci.USA 93: 8374-8378; Ghilardi, N., et al. (1996) Proc. Natl. Acad. Sci.USA 93: 6231-6235). Systemic administration of both CNTF and leptinresulted in induction of tis-11 (Gloaguen, I., et al. (1997) Proc. Natl.Acad. Sci. USA 94: 6456-6461) and STAT3 (Vaisse, C., et al. (1996)Nature Gen. 14: 95-97) in the hypothalamic satiety center, indicatingtheir roles in the regulation of body weight and feeding behavior.Indeed, adminstration of CNTF to humans reduced food intake and resultedin weight loss (Group, A. C. T. S. (1996) Neurology 46:1244-1249.).

SUMMARY OF THE INVENTION

[0023] An object of the present invention is to provide novelCNTF-related neurotrophic factors for the treatment of diseases ordisorders including, but not limited to, motor neuron diseases andmuscle degenerative diseases. In a preferred embodiment, CNTF andrelated molecules are utilized for the treatment of Huntington'sdisease.

[0024] A further object of the present invention is to provide a methodfor identifying CNTF-related factors, other than those specificallydescribed herein, that have improved therapeutic properties.

[0025] These and other objects are achieved in accordance with theinvention, whereby amino acid substitutions in human CNTF proteinenhance its therapeutic properties. In one embodiment, alterations inelectrophoretic mobility are used to initially screen potentially usefulmodified CNTF proteins.

[0026] In a preferred embodiment, the amino acid glutamine in position63 of human CNTF is replaced with arginine (referred to as 63Q→R) oranother amino acid which results in a modified CNTF molecule withimproved biological activity. In further embodiments, rHCNTF variantscombine the 63Q→R mutation with three other novel features:

[0027] 1) Deletion of the last 13 amino acid residues (referred to asΔC13) to confer greater solubility to rHCNTF without impairing itsactivity;

[0028] 2) Substitution of the unique cysteine residue at position 17,which results in stabilization of rHCNTF in physiological buffer, atphysiological pH and temperature conditions without affecting itsactivity; or

[0029] 3) Substitution of amino acid residue 64W, which alters thebiological activity of rHCNTF in vitro and which results in a 7-foldimprovement of its therapeutic index in vivo.

[0030] In another preferred embodiment, a molecule designated RG297(rHCNTF, 17CA63QRΔC13) combines a 63Q→R substitution (which confersgreater biological potency) with a deletion of the terminal 13 aminoacid residues (which confers greater solubility under physiologicalconditions) and a 17CA substitution (which confers stability,particularly under physiological conditions at 37° C.) and shows a 2-3fold better therapeutic index than rHCNTF in an animal model. In anotherpreferred embodiment, a molecule designated RG242 is described thatcarries the double substitution 63QR64WA which results in a differentspectrum of biological potency and a 7-fold higher therapeutic index.

[0031] In another preferred embodiment, a molecule designated RG290 isdescribed that carries the double substitution 63QRΔC13 which confersgreater solubility under physiological conditions.

BRIEF DESCRIPTION OF THE FIGURES

[0032]FIG. 1—Alignment of CNTF protein sequences. A. Human, rat, rabbitmouse and chicken (Leung, et al., 1992, Neuron 8:1045-1053) sequences. 5Dots indicate residues found in the human sequence. Panel B. ModifiedCNTF molecules showing human CNTF amino acid residues (dots) and ratCNTF (residues shown). The name of the purified recombinant proteincorresponding to each sequence is shown on the left.

[0033]FIG. 2—Mobility of human, rat and several modified CNTF moleculeson reducing SDS-15% polyacrylamide gels. Purified recombinant proteinswere loaded as indicated. Markers of the indicated MW were loaded onlane M.

[0034]FIG. 3—Biological activity of two modified CNTF molecules. A.human CNTF (filled diamonds), rat CNTF (open squares), and RPN219(filled squares). B. human CNTF (filled diamonds), rat CNTF (opensquares), and RPN228 (filled squares). Dose response of dissociated E8chick ciliary neurons surviving at the indicated protein concentration,as a percentage of the number of neurons surviving in the presence of 2ng/ml rat CNTF. Each experimental point represents the mean of threedeterminations.

[0035]FIG. 4—Competitive ligand binding towards A.) SCG neurons and B.)MG87/huCNTFR fibroblasts. Standard deviation from the mean of threedeterminations is shown by vertical bars.

[0036]FIG. 5—Mobility of human and several modified CNTF molecules onSDS-15% polyacrylamide gels. Supernatant (A) and pellet (B) preparationsof recombinant human CNTF (designated HCNTF) and several modified CNTFproteins were loaded as indicated. The modified proteins shown are ΔC13(also known as RG160); 17CA,ΔC13 (RG162); ΔC13,63QR (RG290); and17CA,ΔC13,63QR (RG 297). Markers of the indicated MW were loaded on laneM. Incubation in physiological buffer at 37° C. for 0, 2, 7 and 14 daysis indicated in lanes 1-4, respectively.

[0037]FIG. 6—Survival of primary dissociated E8 chick ciliary neurons inresponse to increasing concentrations of various CNTF variants. Controlconcentration response curves for rat CNTF and rHCNTF obtained withstandard, untreated stock solutions, as well as with four rHCNTFvariants, RG297, RG290, RG160 and RG162.

[0038]FIG. 7—Survival of primary dissociated E8 chick ciliary neurons inresponse to increasing concentrations of various CNTF variants. Controlconcentration response curves for rat CNTF and rHCNTF obtained withstandard, untreated stock solutions, as well as with rHCNTF variantRG228 (also known as RPN228 and having the mutation 63QR).

[0039]FIG. 8—Survival of primary dissociated E8 chick ciliary neurons inresponse to increasing concentrations of various CNTF variants. Controlconcentration response curves for rat CNTF and rHCNTF obtained withstandard, untreated stock solutions, as well as with rHCNTF variantRG242 (which has the mutation 63QR,64WA).

[0040]FIG. 9—Average plasma concentration time profiles in the rat afterintravenous (IV) administration of rHCNTF, RG228 and RG242 normalized to100 μg/kg dose for all three compounds.

[0041]FIG. 10—Average plasma concentration time profiles in the ratafter subcutaneous (SC) administration of rHCNTF, RG228 and RG242normalized to 200 μg/kg dose for all three compounds.

[0042]FIG. 11—Comparison of dose dependent rescue of rat muscle wetweight of (A) hCNTF vs. RG228; (B) hCNTF vs. RG297 and (C) hCNTF vs.RG242.

[0043]FIG. 12—Comparison of in vivo toxicity for hCNTF, RG228, RG242 andRG297.

[0044]FIG. 13—Representative Nissl-stained sections (coronal plane) frombrains treated with neurotrophins and injected with quinolinic acid. Topleft: A view of an intact caudate-putamen (CPu). Adjacent panels:Comparable views of sections from brains treated with NGF, BDNF or NT-3and injected with quinolinic acid. In the neurotrophin-treated brains, acircumscribed area (indicated by open arrows) is virtually devoid ofmedium-sized neurons. The two tracks in the CPu were left by theinfusion cannula (c) and the quinolinic acid injection needle(arrowhead). ec, external capsule; LV, lateral ventricle. Scale bar=0.5mm.

[0045]FIG. 14—Representative Nissl-stained sections (coronal plane) frombrains treated with CNTF or PBS and injected with quinolinic acid. Topleft: A view of an untreated, intact caudate-putamen (CPu). Top right: Ahigher magnification view of the lateral CPu showing numerousmedium-sized neurons, a few of which are indicated by arrows. Middle andbottom left: The left CPu in brains treated with PBS or CNTF andinjected with quinolinic acid. The two tracks in the CPu were left bythe PBS or CNTF infusion cannula (c) and the quinolinic acid injectionneedle (arrowhead); open arrows indicate the medial boundary of thelesion. Middle and bottom right: Higher magnification views 250 umlateral to the cannula illustrating the virtually complete absence ofmedium-sized striatal neurons in the PBS-treated brain (neuron lossscore=4), and the presence of numerous, normal-appearing neurons in theCNTF-treated brain (some of the surviving neurons are indicated byarrows; neurons loss score=2). ec, external capsule; LV, lateralventricle. Left scale bar=0.5 mm; right scale bar=30 μm.

[0046]FIG. 15—Effect of treatment with neurotrophic factors onmedium-sized striatal neuron loss induced by intrastriatal injection ofquinolinic acid (QA). A, B, C, D, E. Mean neuron loss scores (±SEM) forgroups treated with neurotrophic factor or PBS and injected withquinolinic acid. The number of rats in each trophic factor-treated groupis as follows: NGF=5; BDNF=12; NT-3=10; CNTF=3; Ax1=7; equivalentnumbers were used in the PBS-treated control groups in each experiment.Statistical comparisons were by unpaired t-test. NT-3 treatment resultedin a significantly greater (+) mean neuron loss score compared with thePBS-treated group: t(17)=2.75, p=0.01. CNTF or Ax1 treatment resulted insignificantly lower (*) mean neuron loss scores compared withPBS-treated groups: t(5)=2.7, p=0.04 and t(13)=4.2, p=0.001,respectively.

[0047]FIG. 16—Effect of treatment with Ax1 on medium-sized striatalneuron loss induced by intrastriatal injection of quinolinic acid (QA).Above each graph, a time line indicates the experimental scheme. A. Meanneuron loss score (γSEM) for groups treated with Ax1 (n=6) or PBS (n=5)in an experimental paradigm similar to that described in FIG. 1 legend,except the osmotic pump was implanted for only 4 days and the injectionof quinolinic acid was given 3 days after removal of the pump. B. Meanneuron loss score (γSEM) for groups receiving a daily intrastriatalinjection of Ax1 (n=6) or PBS (n=6) for 3 days before and 1 day after aninjection of quinolinic acid. *unpaired t-test, A: t(9)=2.5, p=0.03; B:t(10)=2.3, p=0.04.

[0048]FIG. 17—Effects of Axokine-15 (Ax-15) in normal mice. NormalC57BL/6J mice were injected subcutaneously daily for 6 days with eithervehicle or Ax-15 at 0.1 mg/kg, 0.3 mg.kg, or 1.0 mg/kg. Percent changein body weight in Ax-15-treated versus vehicle-treated controls isshown.

[0049]FIG. 18—Effects of Ax-15 in ob/ob mice. C57BL/6J ob/ob mice wereinjected subcutaneously daily for 7 days with either vehicle, leptin(1.0 mg/kg) or Ax-15 at 0.1 mg/kg, 0.3 mg.kg, or 1.0 mg/kg.Diet-restricted, pair-fed mice were injected with 0.3 mg/kg Ax-15 toinvestigate the effects of food intake reduction on weight loss. Percentchange in body weight in Ax-15-treated and leptin-treated versusvehicle-treated controls is shown.

[0050]FIG. 19—Effects of Ax-15 in diet-induced obesity in mice. AKR/Jmice were placed on a high fat diet for seven weeks prior to treatmentwith vehicle, leptin (1.0 mg/kg) or Ax-15 at 0.03 mg/kg, 0.1 mg/kg, 0.3mg/kg, or 1.0 mg/kg. Diet-restricted, pair-fed AKR/J mice were injectedwith 0.3 mg/kg Ax-15 to investigate the effects of food-intake reductionon weight loss. Percent change in body weight in Ax-15-treated andleptin-treated versus vehicle-treated controls is shown.

[0051] FIGS. 20A and 20B—Effects of Ax-15 and diet restriction on seruminsulin and corticosterone levels in diet-induced obese AKR/J mice.

[0052]FIG. 20A—Serum insulin levels were measured in ARK/J diet-inducedobese mice following treatment with vehicle, diet restriction and Ax-15(0.1 mg/kg) or Ax-15 only (0.1 mg/kg) to determine the effects of dietand/or Ax-15 treatment on obesity-associated hyperinsulinemia.

[0053]FIG. 20B—Serum corticosterone levels were measured in ARK/Jdiet-induced obese mice following treatment with vehicle, dietrestriction and Ax-15 (0.1 mg/kg) or Ax-15 only (0.1 mg/kg) to determinethe effects of diet and/or Ax-15 treatment on obesity-associatedhyperinsulinemia.

DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention relates to a method of treatingneurological diseases and disorders in humans or animals. It is based,in part, on the initial finding that recombinant rat CNTF binds moreefficiently to the human CNTF receptor than does recombinant human CNTFand the subsequent discovery that amino acid substitutions which causehuman CNTF to more closely resemble rat CNTF result in enhanced bindingof the modified CNTF to the human CNTF receptor and concomitant enhancedbiological activity.

[0055] In a preferred embodiment, alteration of a single amino acid ofthe human CNTF protein results in a significant enhancement of theability of the protein to promote the survival and outgrowth of ciliaryganglion, as well as other neurons.

[0056] Recombinant human and rat CNTF have the same number of aminoacids (199) and similar mass (MW 22,798 and 22,721 respectively, afterremoval of the N-terminal methionine). Yet, on reducing SDS-PAGE gels,recombinant human CNTF migrates as a protein of MW=27,500, whereas ratCNTF migrates with the expected mobility. In addition, human CNTF hasfour times lower biological activity towards chick ciliary ganglion (CG)neurons than rat CNTF and the human protein competes for binding to thehuman or the rat receptor on cell surfaces much less effectively thanrat CNTF.

[0057] The above observation led to a directed effort to identify theregion on the CNTF molecule responsible for these differences. Thismethod involved the exchange, by genetic engineering methods, of partsof the human CNTF sequence with the corresponding rat CNTF sequence andvice versa. To achieve this, advantage was taken of restriction sitesthat are common to the two CNTF genes and unique in their correspondingexpression vectors. When necessary, such sites were engineered in one orthe other of the two genes in areas that encode the same proteinsequence. With this approach, expression vectors were obtained for eachof the modified proteins shown in FIG. 1. After isolating the individualproteins to at least 60% purity, their properties, as compared to thoseof human and rat CNTF were determined.

[0058] Because the electrophoretic mobilities of human and rat CNTFdiffer significantly, the effect of each amino acid substitution wasmonitored initially by making a determination of the effect of suchchange on the mobility of the protein. As described herein,electrophoretic mobility data indicated that all of the modified humanCNTF molecules that migrated to the same position as rat CNTF had thesingle amino acid substitution Gln63→Arg (Q63→R).

[0059] Modified human CNTF proteins that demonstrated an electrophoreticmobility similar to that of the rat CNTF molecule were subsequentlyexamined for biological activity and receptor binding.

[0060] CNTF is characterized by its capacity to support the survival ofdissociated ciliary neurons of E8 chick embryos. By this criterion,purified recombinant rat CNTF is as active as the native protein fromrat, but four times more active than recombinant human CNTF[Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and inInternational Publication No. WO 91/04316, published on Apr. 4, 1991].The same assay was utilized to determine the biological activity of thealtered molecules prepared as described above. As described herein, allof the modified CNTF molecules that had the Q63→R substitution exhibitedan increased ability to support the survival of ciliary ganglion neuronsas compared to the parent human CNTF protein. Such results indicated astrong correlation between alteration of the electrophoretic mobilityand enhanced biological properties.

[0061] In addition to measuring the biological effect of modificationsmade to human CNTF, an indication of the potential biological activityof each of the molecules may also be obtained by determining the effectof each modification on the ability of the molecules to bind to the CNTFreceptor.

[0062] In one embodiment, the ability of the modified human CNTFproteins to compete with rat CNTF for binding to rat superior cervicalganglia neurons (SCGs) is measured. As described herein, human CNTF isabout 90 times less potent in displacing ¹²⁵I-labelled rat CNTF bindingfrom these cells than unlabelled rat CNTF. Several of the modified humanCNTF proteins described herein, however, are more potent than the humanCNTF in displacing the rat protein. All of the molecules describedherein that had such increased competitive binding ability weremolecules that exhibited altered electrophoretic mobility, wherein themolecules migrated in a manner similar to rat CNTF.

[0063] In another embodiment, cells, such as MG87 fibroblasts, areengineered to express the human CNTF receptor α-component and such cellsare used to assay the binding capability of the modified protein to thehuman receptor. Human CNTF is about 12 times less potent than rat CNTFin competing with ¹²⁵I-labelled rat CNTF for binding to the human CNTFreceptor. Several of the modified human CNTF molecules described herein,including all of those with electrophoretic mobility that resemble ratrather than human CNTF, were more potent than human CNTF in competingwith binding of ¹²⁵I-rat CNTF to the cells expressing the human CNTFreceptor.

[0064] In another embodiment, an animal model with demonstrated utilityin providing an indication of the ability of certain growth and otherfactors to prevent degeneration of retinal photoreceptors may be used toassess the therapeutic properties of the modified CNTF moleculesaccording to the present invention. As described in Example 4, hCNTF(Gln63→Arg) has a ten-fold higher ability than recombinant human CNTF toprevent degeneration of photoreceptors in a light-induced damage modelof retinal degeneration.

[0065] Thus, according to the invention, certain amino acidsubstitutions in the human CNTF protein result in modified human CNTFproteins that exhibit enhanced binding to the human CNTF receptor andtherefore, would be expected to have enhanced therapeutic properties.

[0066] The modified CNTF molecules useful for practicing the presentinvention may be prepared by cloning and expression in a prokaryotic oreukaryotic expression system as described, for example in Masiakowski,et al., 1991, J. Neurosci. 57:1003-1012 and in International PublicationNo. WO 91/04316, published on Apr. 4, 1991. The recombinant neurotrophingene may be expressed and purified utilizing any number of methods. Thegene encoding the factor may be subcloned into a bacterial expressionvector, such as for example, but not by way of limitation, pCP110.

[0067] The recombinant factors may be purified by any technique whichallows for the subsequent formation of a stable, biologically activeprotein. For example, and not by way of limitation, the factors may berecovered from cells either as soluble proteins or as inclusion bodies,from which they may be extracted quantitatively by 8M guanidiniumhydrochloride and dialysis. In order to further purify the factors,conventional ion exchange chromatography, hydrophobic interactionchromatography, reverse phase chromatography or gel filtration may beused.

[0068] According to the present invention, modified CNTF moleculesproduced as described herein, or a hybrid or mutant thereof, may be usedto promote differentiation, proliferation or survival in vitro or invivo of cells that are responsive to CNTF, including cells that expressreceptors of the CNTF/IL-6/LIF receptor family, or any cells thatexpress the appropriate signal transducing component, as described, forexample, in Davis, et al., 1992, Cell 69:1121-1132. Mutants or hybridsmay alternatively antagonize cell differentiation or survival.

[0069] The present invention may be used to treat disorders of any cellresponsive to CNTF or the CNTF/CNTF receptor complex. In preferredembodiments of the invention, disorders of cells that express members ofthe CNTF/IL-6/LIF receptor family may be treated according to thesemethods. Examples of such disorders include but are not limited to thoseinvolving the following cells: leukemia cells, hematopoietic stem cells,megakaryocytes and their progenitors, DA1 cells, osteoclasts,osteoblasts, hepatocytes, adipocytes, kidney epithelial cells, embryonicstem cells, renal mesangial cells, T cells, B cells, etc.

[0070] Accordingly, the present invention provides for methods in whicha patient suffering from a CNTF-related neurological or differentiationdisorder or disease or nerve damage is treated with an effective amountof the modified CNTF, or a hybrid or mutant thereof. The modified CNTFmolecules may be utilized to treat disorders or diseases as describedfor CNTF in International Publication No. WO91/04316 published on Apr.4, 1991 by Masiakowski, et al. and for CNTF/CNTFR complex as describedin International Publication No. WO91/19009 published on Dec. 12, 1991by Davis, et al. both of which are incorporated by reference in theirentirety herein.

[0071] Such diseases or disorders include degenerative diseases, such asretinal degenerations, diseases or disorders involving the spinal cord,cholinergic neurons, hippocampal neurons or diseases or disordersinvolving motorneurons, such as amyotrophic lateral sclerosis or thoseof the facial nerve, such as Bell's palsy. Other diseases or disordersthat may be treated include peripheral neuropathy, Alzheimer's disease,Parkinson's disease, Huntington's chorea (Huntington's disease or HD),or muscle atrophy resulting from, for example, denervation, chronicdisuse, metabolic stress, and nutritional insufficiency or from acondition such as muscular dystrophy syndrome, congenital myopathy,inflammatory disease of muscle, toxic myopathy, nerve trauma, peripheralneuropathy, drug or toxin-induced damage, or motor neuronopathy.

[0072] In one embodiment, CNTF or CNTF-related molecules describedherein are used for the treatment of Huntington's disease. Glutamatereceptor mediated excitotoxicity has been hypothesized to play a role innumerous neurodegenerative diseases or insults, including Huntington'sdisease. The predominant neuropathological feature of Huntington diseaseis a massive degeneration of the medium-sized, GABAergic, striataloutput neurons, without substantial loss of striatal interneurons(Acheson, A. & R. Lindsay., 1994, Seminars Neurosci. 6:333-3410). Asdescribed in Example 7 below, Applicants have conducted studies, usingboth CNTF and the variants described herein, in an animal model whereinthe preferential loss of striatal output neurons observed in Huntingtondisease, and the resulting dyskinesia, are mimicked in rodent or primatemodels in which an NMDA glutamate receptor agonist, quinolinic acid, isinjected into the striatum (DiFiglia, M. Trends Neurosci., 1990,13:286-289). In these studies, CNTF and its variants afforded protectionagainst exposure to quinolinic acid. The close resemblance of theappearance of the quinolinic acid-lesioned striatum to that of patientsdying with HD suggests that quinolinic acid, although it produces anacute and severe lesion in contradistinction to the relentless andrelatively slow progression of HD, constitutes an adequate animal modelfor this devastating neurological disorder.

[0073] To date, human clinical trials using recombinant human CNTF(rHCNTF) have been limited to studies wherein subcutaneousadministration of the protein was tested for its efficacy in slowing theprogression of amyotrophic lateral sclerosis (ALS). Such administrationof rHCNTF was associated with systemic side effects, including coughanorexia and weight loss, and, in at least one study, over 80% ofpatients receiving rHCNTF developed neutralizing antibodies, thesignificance of which is uncertain. However, despite problems with sideeffects and antibody formation, a subgroup of patients in the earlystages of ALS appeared to derive benefit from rHCNTF administration inthat these patients demonstrated a reduced rate of pulmonary functionloss compared to placebo treated patients with similar diseasedurations.

[0074] Preliminary studies conducted by applicants, using intermittent,compartmentalized administration of rHCNTF into the CSF of ALS patients,have demonstrated no evidence of systemic side effects or antibodyformation. Such studies involved the use of an infusion pumpmanufactured by Medtronic (SynchroMed Model 8615/Series DAA) with a sideport for sampling CSF which was implanted under general anesthesia usingstandard techniques (Penn, et al., 1985, 2:125-127). The pump wasattached to a subarachnoid catheter who tip was placed at the L1 levelunder fluoroscopy. Administration of 1 to 8 μg rHCNTF per hour for 48hours each week was tolerated for periods up to 1 year in four patientswith ALS. These patients did not experience the range of adverse eventsseen with systemic rHCNTF administration. Side effects in this patientgroup consisted of sciatic pain in two patients and headaches in one.Elevations in white blood cells and protein were seen in the CSF. Inthis study, rHCNTF displayed similar distribution and pharmacokineticproperties to small molecule drugs such as baclofen and morphine infusedinto the intrathecal space. Unfortunately, rHCNTF is too unstable forcontinuous CNS infusion therapy or for local depot administration, sinceit tends to form covalent dimers through its unpaired cysteine residue,leading to aggregate formation and precipitation. Accordingly, the needexists for stable preparation of CNTF that can be utilized for directinfusion in the central nervous system.

[0075] In collaboration with Aebischer, et al. (unpublished results),Applicants have implanted encapsulated BHK cells which secrete hCNTFinto the subarachnoid space of 10 patients with ALS. Steady-state CSFconcentrations of up to 6 ng/mL have been achieved. Although allpatients complain of asthenia and fatigue, weight loss, anorexia andactivation of the acute phase response proteins were not observed. Therehas been no CSF pleocytosis nor increase in white cell counts. CNTFcannot be detected in the peripheral blood in these patients. Results ofefficacy measures to date are too sparse to permit conclusions regardingefficacy. The lack of an inflammatory response to hCNTF in patientsreceiving rHCNTF synthesized by implanted, encapsulated cells comparedto that seen with pump-infused rHCNTF suggests that the changes seenfollowing pump delivery of rHCNTF may well be related to formulation andstability issues surrounding this particular protein.

[0076] Accordingly, based on animal model data demonstrating theefficacy of CNTF and its variants as protective agents for exitotoxicdamage of striatal neurons in an art recognized model of Huntington'sdisease, combined with Applicants' discovery that the side effects andantibody formation observed using systemic injection of CNTF can beavoided by delivery of CNTF or its variants directly into the CNS,applicants have discovered a useful method of treating Huntington'sdisease. Accordingly, applicants invention contemplates delivery of CNTFor its variants directly into the CNS via implanted cells orcellular-like vesicles, such as, for example, liposomes, which secreteCNTF. Alternatively, CNTF variants as described herein, which haveimproved stability and solubility as compared to CNTF, provide preferredformulations for delivery of CNTF via, for example, osmotic pumps, intothe CNS as described above. Because the instability of rHCNTF insolution at body temperature interferes with its ability to bechronically administered by intrathecal or intraventricular infusion,the variants of rHCNTF described herein are preferred for such uses inview of their improved stability, solubility, and decreasedantigenicity.

[0077] Accordingly, the present invention contemplates variants of CNTFwith improved solubility that may be used in therapeutic applicationswhere infusion, via, for example, osmotic pump, is used to delivery thedrug. The solubility of recombinant human CNTF (rHCNTF) is very limitedin physiological buffer, e.g., Phosphate-Buffered-Saline, pH 7.4 (PBS).Furthermore, the solubility over at least the 4.5-8.0 pH range dependsstrongly on the temperature and on the time of incubation. At 5° C., thesolubility of rHCNTF in PBS is 1 mg/ml and the solution is stable for afew hours, but at 37° C. its solubility is only 0.1 mg/ml after 2 hr and0.05 mg/ml after 48 hrs. This limited solubility and thermal stabilitypreclude stable formulation of rHCNTF in physiological buffer. Suchformulations are particularly desirable for continuous administrationinto the CNS.

[0078] It was discovered that rHCNTF lacking the last 13 amino acidresidues from the carboxyl end (rHCNTF,ΔC13 also designated RPN160 orRG160) retains full biological activity and is soluble at lowtemperatures (5-10° C.) to at least 12 mg/ml. Yet, despite this fargreater solubility, rHCNTF,ΔC13 still falls out of a PBS solution uponincubation at 37° C. over a period of several hours, even atconcentrations as low as 0.1 mg/ml.

[0079] It was determined that the thermal instability of rHCNTF andrHCNTF,ΔC13 was the result of aggregation that was initiated byintermolecular disulfide bond formation and depended strongly on proteinconcentration and temperature. By replacing the single cysteine residueat position 17 of human CNTF with an alanine residue, proteins wereobtained that show far greater stability and maintain their biologicalactivity after incubation for at least 7 days in PBS at 37° C. Thisproperty is maintained in rHCNTF,63QR variants which have higher potencydue to the substitution of the glutamine residue at position 63 byarginine. In a particular example, rHCNTF,17CA,63QR,ΔC13 (alsodesignated RG297) shows greater biological potency than rHCNTF becauseof the 63QR substitution, greater solubility because of the ΔC13deletion and greater stability because of the 17CA substitution.

[0080] The present invention contemplates treatment of a patient havingHD with a therapeutically effective amount of CNTF or the variantsdescribed herein. Effective amounts of CNTF or its variants are amountswhich result in the slowing of the progression of the disease, or of areduction in the side-effects associated with the disease. The efficacyof the treatment may be measured by comparing the effect of thetreatment as compared to controls which receive no treatment. Theclinical course and natural history of HD have been extensivelycharacterized both in field studies (Young et al., 1996, Ann Neurol.20:296-303; Penney and Young, 1990, Movement Disorders 5:93-99), thedevelopment of clinical rating instruments (Shoulson and Fahn, 1979,Neurology 29:1-3; Shoulson et al, 1989, Quantification of NeurologicDeficit, T L Munsat (ed) Butterworths 271-284.; Feigin et al., 1995,Movement Disorders 10:211-214), and radiographic correlates of diseaseprogression using computed X-ray tomography (Terrence et al., 1977,Neuroradiology 13:173-175; Barr et al., 1978, Neurology 28:1196-1200;Neophytides et al., 1979, 23:185-191; Stober et al., 1984,Neuroradiology 26:25-28); magnetic resonance imaging (Grafton et al.,1992, Arch. Neurol. 49:1161-1167) and positron emission tomographictechniques (Harris, et al., 1996, Arch. Neurol. 53:316-324).

[0081] Clinical rating of the progression of Huntington's disease hasbeen assessed using the HD Functional Capacity Scale (HDFC) developed byShoulson and Fahn (1979, Neurology 29:1-3). A fully functional patientreceives a score of 13 on this scale; a score of 0 reflects totaldisability Shoulson et al., 1989, Quantification of Neurologic Deficit,T L Munsat (ed) Butterworths 271-284. The average rate of progression ofpatients using this scale is approximately 0.65 units/year. Shoulson etal., 1989, Quantification of Neurologic Deficit, T L Munsat (ed)Butterworths 271-284; Feigin et al., 1995, Movement Disorders10:211-214. If this scale is truly linear (an hypothesis which has notbeen tested) this rate of progression would correspond nicely with theaverage 20 year duration of symptomatic HD in patients. HDFC scores canbe roughly grouped into 5 clinical stages (Shoulson et al., 1989,Quantification of Neurologic Deficit, T L Munsat (ed) Butterworths271-284).

[0082] Neuroimaging studies have focused on the gross pathologicalconsequences of neuronal loss and consequent atrophy of basal gangliastructures. As HD progresses, the caudate nuclei shrink, giving acharacteristic “box-car” appearance to the lateral ventricles. Thedegree of caudate atrophy can be quantified using a “bicaudate index”.

[0083] Magnetic resonance imaging may be used to generate similarindices to those given by CT. A relatively new technique, in vivo NMRspectroscopy, however, offers the ability to assess metabolic processeswithin the living brain. One preliminary study (Jenkins, et al., 1993,Neurology 43:2689-2695 has detected an increased amount of lactic acid,presumably reflecting either neuronal cell loss or a defect inintermediary metabolism, in the brains of HD patients.

[0084] Positron Emission tomographic (PET) permits functional imaging tobe performed in living patients. Changes in metabolic state can beassessed using 2-deoxyglucose (which reflects synaptic activity), orselective radioligands which mark selected neuronal populations. Todetermine the rate of change of glucose metabolism and caudate size inpersons at risk for Huntington's disease, Grafton et al., (1992, ArchNeurol. 49:1161-1167) evaluated 18 persons at risk for Huntington'sdisease with two positron emission tomographic glucose metabolic studiesand two magnetic resonance imaging scans separated by 42 (+/−9) months.Seven of the individuals were Huntington' disease gene negative; theremainder were gene positive by genetic testing or onset of chorea afterstudy entry. The gene-positive group demonstrated a significant 3.1%loss of glucose metabolic rate per year in the caudate nucleus (95%confidence interval [CI], −4.64, −1.48) compared with the gene-negativegroup. There was a 3.6% per year increase in the magnetic resonanceimaging bicaudate ratio (95% CI, 1.81, 5.37), a linear measure ofcaudate atrophy. However, the rate of change in caudate size did notcorrelate with the rate of change in caudate metabolism, suggesting thatmetabolic loss and atrophy may develop independently. Thus serialpositron emission tomographic or magnetic resonance imaging yield ratesof loss not too different from those observed in clinical rating scales(approximately 5% per year, vide supra), and thus may be useful means bywhich to monitor experimental pharmacologic interventions inpresymptomatic individuals at risk for HD should clinical trials bedesigned to incorporate such a patient population.

[0085] In addition to glucose metabolic mapping, other radioligands maybe used to monitor striatal integrity in HD. For example, sinceintrinsic striatal neurons which are lost in HD uniformly bear dopaminereceptors, ligands for the dopamine receptor have been used to monitorthe progression of HD. These studies do indeed show a parallel reductionof both striatal D1 and D2 receptors in HD patients (Turjanski et al.,1995, Brain 118:689-696).

[0086] Similar metabolic and neurochemical findings have been obtainedin PET studies of primates treated with quinolinic acid in the striatum.Brownell et al., (1994, Exp. Neurol. 125:41-51), reported that,following a quinolinate lesion of the striata of 3 non-human primates,symptoms similar to those of Huntington's disease could be induced bydopamine agonist treatment. All animals showed a long-term 40-50%decrease in glucose utilization in the caudate by[19F]fluoro-2-deoxy-D-glucose positron emission tomography (PET).Caudate-putamen uptake rate constants for Dl receptors reflectedneuronal loss and decreased by an average 40 to 48%. Dopamine reuptakesites and fibers assessed by PET showed a temporary decrease in areaswith mild neuronal loss and a long-term decrease in striatal regionswith severe destruction. These results, which were consistent withbehavioral changes and neuropathology seen at postmortem examination,are similar to those observed in clinical studies of Huntington'sdisease patients, and serve to additionally validate the quinolinic acidmodel, and suggest that these measures may be of use in human clinicaltrials.

[0087] Clinical trials in HD have largely been limited to the assessmentof palliative symptomatic therapies for psychiatric symptoms andinvoluntary movements (Shoulson et al., 1981, Neurology 29:1-3).However, there has been one attempt to examine a potentialneuroprotective agent. This trial involved the use of baclofen, a GABA-Breceptor antagonist, on the theory that this agent would reduceglutamate release from corticostriatal terminals in the striatum,thereby retarding the progression of HD (Shoulson et al., 1989,Quantification of Neurologic Deficit, T L Munsat (ed) Butterworths271-284). The outcome of this trial was negative, in thatbaclofen-treated patients fared no better than controls over the30-month duration of the trial. Nonetheless, this trial provided theproving ground for the use and validation of the HDFC. One importantoutcome of the study was that the intrinsic rate of disease progressionin the study subjects was only one-half of that originally estimated bythe investigators. This information may now be used in the design offuture clinical trials using this rating instrument.

[0088] Currently, there are no major ongoing clinical trials in HD.However, a clinical trials organization, the Huntington's disease StudyGroup, has been organized and has the infrastructure in place for theconduct of clinical trials in HD. This group is currently investigatinga variety of clinical trial options including 1) the use of Coenzyme Qto enhance intermediary metabolism and 2) the use of glutamateantagonists and/or glutamate release blockers (W. Koroshetz, personalcommunication). A parallel group has been established in Europe, andthis group will be using PET methodology to examine the potentialefficacy of fetal striatal implants and, eventually, the use ofxenograft transplants as well.

[0089] The availability of a validated clinical rating instrument, andthe existence of correlative radiographic measures to assess diseaseprogression in HD, combined with the existence of 2 large, organizedmulticenter clinical trials consortia will make implementation ofclinical trials in HD straightforward.

[0090] Applicants describe herein the production of a modified CNTFmolecule, known as Ax-13 or Ax-1, (designated rHCNTF,17CA63QRΔC13) whichcombines a 63Q→R substitution (which confers greater biological potency)with a deletion of the terminal 13 amino acid residues (which confersgreater solubility under physiological conditions) and a 17CAsubstitution (which confers stability, particularly under physiologicalconditions at 37° C.) and shows a 2-3 fold better therapeutic index thanrHCNTF in an animal model. However, when expressed in E. coli, asubstantial portion of the expressed protein produced is tagged with adecapeptide at the C-terminus. Because of this, purification of Ax-13 isdifficult and results in a low yield of purified, untagged product. Thisdecapeptide tagging likely does not occur when the Ax-13 is expressed ina mammalian expression system. In addition, it is possible that thedecapeptide tag could contribute to increased immunogenicity of themolecule and may also possibly cause problems with stability.

[0091] However, use of the E. coli expression system would be preferablefrom the standpoint of cost and efficiency. Therefore, applicantsundertook to develop a truncated CNTF molecule that would retain theimproved potency, solubility and stability properties of Ax-13, whileavoiding the problem of decapeptide tagging when expressed in E. coli.As described herein, applicants have succeeded in producing a moleculeknown as Ax-15, (designated rHCNTF,17CA63QRΔC15), which retains theimproved properties of Ax-13, but which also has the added advantage ofbeing expressed by E. coli with reduced amino acid tag being added. Thenew molecule, Ax-15, therefore has the advantage of being more easilypurified with a greater yield. Additionally, because there is greatlyreduced bacterial amino acid tagging, Ax-15 does not raise the concernwith regard to the immunogenicity or stability of the molecule thatcould be raised by Ax-13.

[0092] Therefore the object of the present invention is to provide animproved modified ciliary neurotrophic factor molecule. Specifically,one embodiment of the invention is a modified human ciliary neurotrophicfactor having the modification Cys17→Ala, Gln63→Arg, and a deletion ofthe terminally amino acid residues. The present invention also providesfor an isolated nucleic acid molecule encoding the modified humanciliary neurotrophic factor of the invention. Also, contemplated by theinvention is a recombinant DNA molecule that encodes the modified humanciliary neurotrophic factor of the invention and which is operativelylinked to an expression control sequence, as well as a host celltransformed with the recombinant DNA molecule. The host cell may beprokaryotic or eukaryotic, and therefore may be, for example, abacterium such as E. coli, a yeast cell such as Pichia pastoris, aninsect cell such as Spodoptera frugiperda, or a mammalian cell such as aCOS or CHO cell. Said host cell may be used in a method for producingthe modified ciliary neurotrophic factor molecule comprising: (a)growing the host cell transformed with the recombinant DNA molecule ofthe invention so that the DNA molecule is expressed by the host cell toproduce the modified ciliary neurotrophic factor molecule of theinvention and (b) isolating the expressed, modified ciliary neurotrophicfactor molecule.

[0093] The subject invention further contemplates a compositioncomprising the modified ciliary neurotrophic factor molecule of theinvention (Ax-15), and a carrier.

[0094] Another object of the present invention is to provide a method oftreating a disease or disorder of the nervous system comprisingadministering the modified ciliary neurotrophic factor described hereinas Ax-15. The disease or disorder treated may be a degenerative diseaseand/or involve the spinal cord, motor neurons, cholinergic neurons orcells of the hippocampus. Alternatively, the method of treatment may befor treating a disease or disorder of the nervous system which comprisesdamage to the nervous system caused by an event selected from the groupconsisting of trauma, surgery, infarction, infection, malignancy andexposure to a toxic agent. Also contemplated by the present invention isa method of treating a disease or disorder involving muscle atrophy.

[0095] A further object of the present invention is to provide a methodof protecting striatal neurons from degeneration comprising treatingsaid striatal neurons with an effective amount of the modified ciliaryneurotrophic factor described herein as Ax-15.

[0096] Also contemplated by the present invention is a method oftreating Huntington's disease comprising direct administration to thecentral nervous system of the modified ciliary neurotrophic factordescribed herein-as Ax-15.

[0097] A further object of the present invention is to provide a methodof inducing weight loss in a mammal comprising administration to themammal of the modified ciliary neurotrophic factor described herein asAx-15. A specific embodiment of this invention involves inducing weightloss in a human.

[0098] The method of administering Ax-15 may be used in the treatment ofmorbid obesity or obesity of a genetically determined origin. The Ax-15described herein may also be used in a method of preventing and/ortreating the occurrence of gestational or adult onset diabetes in ahuman.

[0099] Any of the above-described methods involving the administrationof Ax-15 may be practiced by administering the Ax-15 via a route ofdelivery selected from the group consisting of intravenous,intramuscular, subcutaneous, intrathecal, intracerebroventricular andintraparenchymal.

[0100] Alternatively, the Ax-15 may be administered via the implantationof cells that release the modified ciliary neurotrophic factor.

[0101] The present invention also contemplates diseases or disordersresulting from damage to the nervous system, wherein such damage may becaused by trauma, surgery, infarction, infection and malignancy or byexposure to a toxic agent.

[0102] The present invention also provides for pharmaceuticalcompositions comprising a modified CNTF molecule or hybrid or mutantthereof, as described herein, as the sole therapeutic agent or in acomplex with the CNTF receptor, in a suitable pharmacologic carrier.

[0103] The active ingredient, which may comprise CNTF or the modifiedCNTF molecules described herein should be formulated in a suitablepharmaceutical carrier for administration in vivo by any appropriateroute including, but not limited to intraparenchymal, intraventricularor intracerebroventricular delivery, or by a sustained release implant,including a cellular or tissue implant such as is described, forexample, in published application WO96/02646 published on Feb. 1, 1996,WO95/28166 published on Oct. 26, 1995, or WO95/505452 published Feb. 23,1995.

[0104] Depending upon the mode of administration, the active ingredientmay be formulated in a liquid carrier such as saline, incorporated intoliposomes, microcapsules, polymer or wax-based and controlled releasepreparations, In preferred embodiments, modified CNTF preparations whichare stable, or formulated into tablet, pill or capsule forms.

[0105] The concentration of the active ingredient used in theformulation will depend upon the effective dose required and the mode ofadministration used. The dose used should be sufficient to achievecirculating plasma concentrations of active ingredient that areefficacious. Effective 20 doses may be extrapolated from dose-responsecurves derived from in vitro or animal model test systems. Effectivedoses are expected to be within the range of from about 0.001 to about 1mg/day.

EXAMPLES Example 1 Electrophoretic Mobility of Modified Human CNTFMolecules

[0106] Materials and Methods

[0107] Preparation of Modified CNTF Molecules

[0108] Bacterial Strains and Plasmids

[0109]E. coli K-12 RFJ26 is a strain that overproduces the lactoseoperon repressor.

[0110] The expression vectors pRPN33, which carries the human CNTF geneand pRPN110 which carries the rat CNTF gene are nearly identical(Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and inInternational Publication No. WO 91/04316, published on Apr. 4, 1991.)

[0111] Plasmid pRPN219 was constructed by first digesting pRPN33 withthe restriction enzymes NheI plus Hind3 and gel purifying the 4,081 bpfragment. The second, much smaller fragment which codes for part of thehuman CNTF gene was subsequently replaced with an 167 bp NheI-Hind3fragment that was obtained by PCR amplification from the rat gene usingthe primers RAT-III-dniH: 5′ ACGGTAAGCT TGGAGGTTCTC 3′; and RAT-Nhe-1-M:5′ TCTATCTGGC TAGCAAGGAA GATTCGTTCA GACCTGACTG CTCTTACG 3′.

[0112] Plasmid pRPN228 was constructed in the same manner as pRPN219,except that the 167 bp replacement fragment was amplified using the DNAprimers Rat-III-dniH-L-R: 5′ AAG GTA CGA TAA GCT TGG AGG TTC TCT TGG AGTCGC TCT GCC TCA GTC AGC TCA CTC CAA CGA TCA GTG 3′ and Rat-Nhe-1: 5′ TCTATC TGG CTA GCA AGG AAG 3′.

[0113] Plasmids pRPN186, pRPN187, pRPN188, pRPN189, pRPN192, pRPN218,and pRPN222 were generated by similar means or by direct exchange of DNAfragments using the unique restriction sites shown in FIG. 1.

[0114] The identity of all plasmids was confirmed by restrictionanalysis and DNA sequencing.

[0115] Protein Purification

[0116] Induction of protein synthesis, selective extraction, andpurification from inclusion bodies were as described for rat and humanCNTF (Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and inInternational Publication No. WO 91/04316, published on Apr. 4, 1991)except that gel filtration was occasionally used instead or in additionto ion exchange chromatography. Alternatively, proteins were purifiedfrom the supernatants of cell lysates by streptomycin and ammoniumsulfate fractionation, followed by column chromatography, as describedfor other proteins (Panayotatos et al., 1989, J. Biol. Chem.264:15066-15069). All proteins were isolated to at least 60% purity.

[0117] Conditions for enzymatic reactions, DNA electrophoresis and othertechniques used in these studies have been described in detail(Panayotatos, N. 1987, Engineering an Efficient Expression System inPlasmids: A practical Approach (Hardy, K. G. ed.) pp 163-176, IRL Press,Oxford, U.K.).

[0118] Results

[0119] The mobilities of human, rat and several chimeric CNTF moleculeson reducing SDS-polyacrylamide gels are shown in FIG. 2. The chimericmolecules RPN186, RPN189, RPN218 and RPN228 exhibit mobilitiescomparable to rat CNTF, whereas RPN187, RPN188, RPN192 and RPN222exhibit mobilities comparable to human CNTF. Cross-reference of theseresults to the aligned sequences of these proteins in FIG. 1 revealsthat all proteins carrying an arginine residue at position 63 (R63)display the mobility of rat CNTF. In the case of RPN228, this singleamino acid substitution (Q63->R) is sufficient to confer to human CNTFthe normal mobility of rat CNTF.

[0120]FIG. 2 also provides a measure of the purity of the differentrecombinant proteins. By visual inspection, purity varies from 60% forRPN189 to better than 90% for RPN228.

Example 2 Measurement of Binding Activity of Modified CNTF Molecules

[0121] Materials and Methods

[0122] Preparation of 125I-CNTF

[0123] Recombinant rat CNTF (28 μg) in 37 μl 0.2 M sodium borate buffer,pH 8.5 was transferred to a vial containing 4 mCi, (2,000 Ci/mmole; NEN)of ¹²⁵I and reagent (Bolton and Hunter, 1973, Biochem J. 133: 529-539)which had been dried under a gentle stream of nitrogen. Reactions wereincubated for 45 min at 0° C. followed by 15 min at room temperature andterminated by the addition of 30 ml of 0.2 M glycine solution. After 15min, 0.2 ml PBS containing 0.08% gelatin was also added and the mixturewas passed through a Superdex-75 column (Pharmacia) to separate thelabelled monomeric CNTF from dimeric and other multimeric derivatives.Percentage of incorporation was typically 20%, as determined by thinlayer chromatography and the specific activity was typically around1,000 Ci/mmole. The monomeric ¹²⁵I-CNTF was stored at 4° C. and used upto one week after preparation. As a test of structural andconformational integrity, ¹²⁵I-CNTF (approximately 10,000 cpm) was mixedwith a 5 μg unlabelled CNTF and analyzed by native gel electrophoresis.One major band was visible by either Coomassie staining orautoradiography. ¹²⁵I-CNTF also showed comparable activity to nativeCNTF in supporting survival of E8 chick ciliary neurons in culture.

[0124] Tissue Culture Techniques

[0125] Superior cervical ganglia (SCG) from neonatal rats were treatedwith trypsin (0.1%), mechanically dissociated and plated on apoly-ornithine (30 μg/ml) substratum. Growth medium consisted of Ham'snutrient mixture F12 with 10% heat-inactivated fetal bovine serum(Hyclone), nerve growth factor (NGF) (100 ng/ml), penicillin (50 U/ml)and streptomycin (50 μg/ml). Cultures were maintained at 37° C. in ahumidified 95% air/5% CO₂ atmosphere. Ganglion non-neuronal cells wereeliminated by treatment with araC (10 μM) on days 1 and 3 of culture.Cultures were fed 3 times/week and were routinely used for bindingassays within 2 weeks.

[0126] MG87/CNTFR is a fibroblast cell line transfected with the humanCNTFα receptor gene (Squinto, et al., 1990, Neuron 5:757-766; Davis etal., 1991, Science 253:59-63).

[0127] Binding Assays

[0128] Binding was performed directly on cell monolayers. Cells inculture wells were washed once with assay buffer consisting of phosphatebuffered saline (PBS; pH 7.4), 0.1 mM bacitracin, 1 mM PMSF, 1 μg/mlleupeptin, and 1 mg/ml BSA. After incubation with ¹²⁵I-CNTF for 2 hoursat room temperature, cells were quickly washed twice with assay buffer,lysed with PBS containing 1% SDS and counted in a Packard Gamma Counter.Non-specific binding was determined in the presence of 1,000-fold excessof unlabelled CNTF. Specific binding towards MG87/CNTFR was 80-90%. Datawere analyzed using the GRAPHPAD program (ISI, Philadelphia, Pa.).

[0129] Results

[0130] Competition curves of purified recombinant human, rat and CNTFRPN219 towards ¹²⁵I-rat CNTF for binding on rat SCG neurons are shown inFIG. 4a. Both rat and human CNTF compete with ¹²⁵I-rat CNTF for bindingto SCG neurons, but human CNTF (IC50=25 nM) is 90 times less potent indisplacing ¹²⁵I-rat CNTF binding than unlabelled rat CNTF (IC50=0.28nM). In contrast, RPN219 is almost as potent as rat CNTF and clearlymore potent than human CNTF (IC50=0.3 nM). Similar results were obtainedfrom competition experiments with mouse fibroblasts transfected with aplasmid directing the expression of the human CNTF receptor (FIG. 4b).Both rat, human and RPN228 compete with ¹²⁵I-rat CNTF for binding toMG87/CNTFR cells. Human CNTF (IC50=30 nM) is 12 times less potent thanrat CNTF (IC50=2.8 nM), whereas RPN228 is clearly more potent than thehuman protein (IC50=5.6 nM).

[0131] Competition binding experiments with the other modified CNTFproteins shown in FIG. 1 also demonstrated that proteins having R63displayed the biological activity of rat CNTF, whereas proteins having063 displayed the binding properties of human CNTF (data not shown).These results indicate that the single amino acid substitution (063->R)is sufficient to confer to human CNTF the receptor binding propertiescharacteristic of rat CNTF.

Example 3 Measurement of Biological activity of Modified CNTF Molecules

[0132] Materials and Methods

[0133] Recombinant CNTF was assayed on dissociated cultures of chickciliary ganglion (CG) neurons as described (Masiakowski, et al., 1991,J. Neurosci. 57:1003-1012 and in International Publication No. WO91/04316, published on Apr. 4, 1991), except that surviving cells werestained with MTT (Mosmann, T. 1983; J. Immunol. Methods 65:55-63).

[0134] Results

[0135]FIG. 3 shows dose-response curves of dissociated, neuron-enrichedcultures of E8 chick embryo ciliary ganglia for purified recombinanthuman, rat and the modified CNTF proteins RPN219 and RPN228. By thisassay, the biological activity of the chimeric proteins isindistinguishable from that of purified recombinant rat CNTF and clearlyhigher than that of recombinant human CNTF. Comparison of thedose-response curves in FIG. 3 also shows that the maximal levels ofsurviving neurons obtained with RPN219, RPN228 or rat CNTF are higherthan those obtained with human CNTF. These results suggest that RPN219and RPN228, like rat CNTF, are active towards a larger population ofneurons than human CNTF. In parallel experiments, the biologicalactivity of the other modified CNTF proteins shown in FIG. 1 wasexamined. In every case, modified CNTF proteins carrying the (Q63→R)substitution displayed the biological activity of rat CNTF whereasproteins having Q63 displayed the activity of human CNTF (data notshown).

[0136] Overall, these results indicate that the single amino acidsubstitution (Q63→R) is sufficient to confer to human CNTF thebiological activity of rat CNTF.

Example 4 Use of Modified CNTF to Prevent Light Induced PhotoreceptorInjury

[0137] Albino rats of either the F344 or Sprague-Dawley strain were usedat 2-5 months of age. The rats were maintained in a cyclic lightenvironment (12 hr on: 12 hr off at an in-cage illuminance of less than25 ft-c) for 9 or more days before being exposed to constant light. Therats were exposed to 1 or 2 weeks of constant light at an illuminancelevel of 115-200 ft-c (most rats received 125-170 ft-c) provided by two40 watt General Electric “cool-white” fluorescent bulbs with a whitereflector that was suspended 60 cm above the floor of the cage. Duringlight exposure, rats were maintained in transparent polycarbonate cageswith stainless steel wire-bar covers.

[0138] Two days before constant light exposure, rats anesthetized with aketamine-xylazine mixture were injected intravitreally with 1 μl of ratCNTF, human CNTF or modified CNTF [hCNTF (Q63→R)] dissolved in phosphatebuffered saline (PBS) at a concentration of 0.1 to 500 ng/μl. Theinjections were made with the insertion of a 32 gauge needle through thesclera, choroid and retina approximately midway between the ora serrataand equator of the eye. In all cases, the injections were made into thesuperior hemisphere of the eye.

[0139] Immediately following constant light exposure, the rats weresacrificed by overdose of carbon dioxide followed immediately byvascular perfusion of mixed aldehydes. The eyes were embedded in epoxyresin for sectioning at 1 μm thickness to provide sections of the entireretina along the vertical meridian of the eye. The degree oflight-induced retinal degeneration was quantified by assessing thedegree of photoreceptor rescue by a 0-4+ pathologist's scale of rescue,4+ being maximal rescue and almost normal retinal integrity. The degreeof photoreceptor rescue in each section, as based on comparison to thecontrol eye in the same rat, was scored by four individuals. This methodhas the advantage of considering not only the ONL thickness, but alsomore subtle degenerative changes to the photoreceptor inner and outersegments, as well as spatial degenerative gradients within the eye.Three eyes were examined for each time point to generate a dose responsecurve.

[0140] Results

[0141] The degree of rescue was measured for human, rat and hCNTF(Q63→R). The data indicated that both rat and hCNTF (Q63→R) had ten-foldgreater ability to rescue photoreceptors in the light damage model thandid recombinant human CNTF.

[0142] It is to be understood that while the invention has beendescribed above in conjunction with preferred specific embodiments, thedescription and examples are intended to illustrate and not limit thescope of the invention, which is defined by the scope of the appendedclaims.

Example 5

[0143] Materials and Methods

[0144] Recombinant human CNTF variants were genetically engineered,expressed in E. coli and recovered at greater than 90% purity, asdescribed previously (Masiakowski, et al., 1991, J. Neurosci.57:1003-1012 and in International Publication No. WO 91/04316, publishedon Apr. 4, 1991; Panayotatos et al., 1993, J. Biol. Chem.268:19000-19003).

[0145] The following stock solutions were prepared freshly in PBS at 5°C.: rHCNTF 0.5 mg/ml RG160 (rHCNTF,ΔC13) 0.5 mg/ml RG162(rHCNTF,17CA,ΔC13) 0.5 mg/ml RG290 (rHCNTF,63QR,ΔC13) 1.2 mg/ml RG297(rHCNTF,17CA,63QR,ΔC13) 0.4 mg/ml

[0146] To determine the stability of rHCNTF and several derivatives inphysiological buffer at 37° C., stock solutions were dialyzedexhaustively against PBS at 5° C., diluted with PBS to 0.1 mg/ml andsterilized by filtration. Aliquots (0.2 ml), were transferred into 0.5ml capacity polypropylene centrifugation tubes. The tubes were placed ina 37° C. incubator and, at the indicated times, individual tubes wereremoved and centrifuged at 15,000 rpm for 3 min at room temperature toseparate soluble protein from insoluble precipitates. Supernatants werepipetted off into clean tubes containing an equal volume of 2×proteingel sample buffer, placed in a 85° C. bath for 2 min, mixed and storedat −20° C. until analysis by 15% SDS-PAGE. Pellets were resuspended in{fraction (1/10)} original volume of water, mixed with an equal volumeof 2×protein gel sample buffer and treated as above.

[0147] Methods for biological activity assays on E8 chick ciliaryneurons and for protein gel electrophoresis have been described(Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and inInternational Publication No. WO 91/04316, published on Apr. 4, 1991;Panayotatos et al., 1993, J. Biol. Chem. 268:19000-19003). Protein gelsample buffer (2×) consists of 12.5 ml Tris HCl, pH 6.8—20 mlglycerol—40 ml 10% SDS and 5 mg Bromophenol Blue per 100 ml.

[0148] Results

[0149] The solubility of rHCNTF is particularly limited in physiologicalbuffer at neutral pH. Furthermore, the solubility over a broad pH range(4.5-8.0) depends strongly on the temperature and on the time ofincubation. At 5° C., the solubility of rHCNTF in PBS is 1.4 mg/ml andthe protein remains in solution for a few hours. In sharp contrast tothe limited solubility of rHCNTF, the variant rHCNTF,ΔC13 can beconcentrated to at least 12 mg/ml at 5° C. Despite this greatersolubility, however, rHCNTF,ΔC13 still shows strong instability inphysiological buffer, pH and temperature conditions. Upon incubation at37° C., rHCNTF,ΔC13 falls out of solution at a rate that depends on theinitial concentration.

[0150] To determine the cause of this instability, we analyzed thephysical integrity of rHCNTF and several variants in parallelexperiments. FIG. 5 shows that incubation of rHCNTF in physiologicalbuffer at 37° C. for 0, 2, 7 and 14 days (lanes 1-4, respectively)caused progressive disappearance of the protein from the supernatants,accompanied by concomitant progressive appearance in the pellets.Furthermore, a good proportion of rHCNTF in the pellets appeared as a 48kD species that corresponded to the size of dimeric rHCNTF (FIG. 5,double arrow). At longer incubation times, a small proportion of higherorder aggregates was also evident. However, when the same samples wereanalyzed on the same type of gel but in the presence of disulfidereducing agents, the 48 kD species was converted to monomeric rHCNTF,evidence that the 48 kD species represents rHCNTF dimers covalentlylinked by disulfide bonds. Such dimers would be expected to form throughthe unique cysteine residue of rHCNTF. Therefore, these resultsindicated that the instability of rHCNTF at 37° C. is caused byaggregation initiated by intermolecular disulfide bond formation.

[0151] Similar results were obtained with two rHCNTF variants,rHCNTF,ΔC13 and rHCNTF,63QR,ΔC13, except that the appearance ofinsoluble aggregates in the pellets was somehow slower in the case ofrHCNTF,ΔC13 (FIG. 5). Given the fact that the ΔC13 deletion confers torHCNTF much greater solubility in physiological buffer, the improvedstability of rHCNTF,ΔC13 is most likely an indirect consequence of itsgreater solubility.

[0152] To further test the possibility that the instability of rHCNTF at37° C. is caused by aggregation initiated by intermolecular disulfidebond formation, the unique cysteine residue at position 17 wassubstituted by alanine, using established genetic engineeringmethodology. The two rHCNTF variants, rHCNTF,17CA,ΔC13 andrHCNTF,17CA,63QR,ΔC13 generated by this process were subjected to thesame analysis by non-reducing 15% SDS-PAGE. FIG. 5 shows that even afterincubation for 14 days at 37° C. both proteins remained soluble with noevidence of dimerization or aggregate formation. Even in the smallproportion of protein found in the pellets, which represented mostly thesmall amount of soluble protein remaining in the centrifuge tubes afterremoval of the supernatant, there was little evidence of dimerization.These results confirmed the conclusion that the instability of rHCNTF iscaused by aggregation initiated by intermolecular disulfide bondformation, and demonstrated that elimination of the free —SH functionalgroup in other rHCNTF variants, e.g. RG297, also result in greaterstability.

[0153] To test whether the proteins remaining in solution afterincubation at 37° C. were still biologically active, samples wereanalyzed for neuronal survival activity. FIG. 6 shows controlconcentration response curves for rat CNTF and rHCNTF obtained withstandard, untreated stock solutions, as well as with four rHCNTFvariants incubated for 7 days at 37° C. Of the latter, the proteinscarrying the 17CA mutation, RG297 and RG162, were assayed at theirnominal concentrations, whereas RG290 and RG160 were assayed aftercorrecting their concentrations for the amount of protein remaining insolution. FIG. 6 shows that the concentration response curves displayedby these compounds are those expected from these proteins in their fullyactive form: RG160 and RG162 show the same potency as rHCNTF withinexperimental error, whereas RG290 and RG297 that carry the 63QRsubstitution show 4-5 fold higher potency than rHCNTF, as previouslyobserved (Panayotatos, N., et al., 1993, J. Biol. Chem. 268:19000-19003)and as shown in FIG. 7. Therefore, incubation of rHCNTF and itsderivatives at 37° C. for 7 days does not cause loss of biologicalactivity, only loss of protein through dimerization followed byprecipitation.

Example 6

[0154] Materials and Methods

[0155] Protein Engineering and Purification—The following rHCNTFvariants were compared to rHCNTF:

[0156] RG228 (rHCNTF,63QR);

[0157] RG297 (rHCNTF,17CA,63QR,ΔC13)

[0158] RG242 (rHCNTF,63QR64WA)

[0159] These proteins were genetically engineered, expressed in E. coliand recovered at greater than 90% purity by the methodology describedfor rHCNTF (Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and inInternational Publication No. WO 91/04316, published on Apr. 4, 1991;Panayotatos et al., 1993, J. Biol. Chem. 268:19000-19003).

[0160] Biological Activity Assays—Methods for biological activity assayson E8 chick ciliary neurons have been described (Panayotatos et al.,1993, J. Biol. Chem. 268:19000-19003).

[0161] Pharmacokinetic Determinations—Rats were injected intravenously(i.v.) with rHCNTF (n=1) and RG242 (n=2) at 100 μg/kg and with RG228(n=1) at 200 ug/kg. Rats were also injected subcutaneously (s.c.) withrHCNTF (n=2), RG242 (n=2) and RG228 (n=1) at 200 ug/kg. Blood specimenswere collected prior to dosing and at various times after dosing andwere processed to obtain plasma. The plasma specimens were analyzedusing the rHCNTF ELISA method for rodent plasma (D. B. Lakings, et al.DSER 93/DMAP/006, “Dose Proportionality and Absolute Bioavailability ofrHCNTF in the Rat Following Subcutaneous Administration at Eight DoseLevels” (Phoenix International Project No. 920847) Nov. 10, 1993).

[0162] The plasma concentrations were evaluated using non compartmenttechniques. A standard curve for each compound was included on eachassay plate and was used to calculate the amount of that compoundpresent in the specimens analyzed on the plate. The sensitivity of theassay varied among compounds by less than twofold.

[0163] Efficacy and Toxicity Determinations In Vivo—Male Sprague-Dawleyrats weighing μ220 g were anesthetized before surgery. The right sciaticnerve was transected at the level of the knee and a 5 mm segment ofnerve was removed. Sham surgeries were performed on the left side ofeach animal. Starting the morning after surgery, rats were weighed andadministered vehicle (either PBS or lactate/phosphate/mannitol, pH 4.5)or the rHCNTF compound to be tested, dissolved in the same vehicle atdoses ranging from 0.01-1.0 mg/kg, s.c. Rats were weighed and injecteddaily for 1 week, at which time they were sacrificed and the soleusmuscles dissected and weighed. The ratio of the right (denervated) toleft (sham) soleus wet weights for each animal was calculated to assessthe degree of atrophy caused by denervation and the prevention thereofby treatment with each compound. For assessment of toxicity, the bodyweights were calculated as a percent of the weight gain ofvehicle-treated rats. Both vehicle solutions produced similar results inatrophy and body weight gain.

[0164] Results

[0165] Biological Activity In vitro—To characterize the activity ofrHCNTF in vitro, we measured its effect on mediating the survival ofprimary dissociated E8 chick ciliary neurons. Neuronal survival inresponse to increasing concentrations of various human CNTF variants isshown in FIGS. 6, 7 and 8. The variants RG228 (FIG. 7) and RG297 (FIG.8) that carry the 63QR substitution show 4-5 times greater potency thanrHCNTF but the variant RG242 showed a 10-fold weaker potency thanrHCNTF, despite the fact that it carries the 63QR substitution. Thus,introduction of various amino acid side chains at various positions ofthe CNTF sequence has very different effects on the survival of primaryneurons in vitro that vary from great loss to strong gain of activityrelative to rHCNTF.

[0166] Pharmacokinetics—Before attempting to correlate the in vitrobiological potency of a set of compounds to their pharmacologicalefficacy in vivo, it is useful to determine their absolutebioavailability in the same animal model. In the experiments describedbelow, the disposition kinetics after i.v. administration and theabsolute bioavailability after s.c. administration of RG228 and RG242were determined and compared to those of rHCNTF.

[0167] The average plasma concentration time profiles in the rat afterIV administration of rHCNTF, RG228 and RG242 are shown in FIG. 9,normalized to 100 μg/kg dose for all three compounds. The averagepharmacokinetic parameters are summarized in Table 1.

[0168] After i.v. administration to rats, RG242 had a distribution phaseα somewhat faster than that of rHCNTF and RG228. The disposition phase βfor RG242 and RG228 was faster than that of rHCNTF. Thus, RG242 appearedto be distributed into the body and cleared from systemic circulationsomewhat more rapidly than rHCNTF, whereas RG228 appeared to bedistributed into the body as fast as rHCNTF and cleared from systemiccirculation somewhat faster. The area under the concentration time curve(AUC) for RG242 was comparable to that of rHCNTF, indicating that thetotal body clearance (CI_(T)) was about the same for the two compounds.A twice larger area was observed with RG228. However, the apparentvolume of distribution (V_(area)), which is a function of both β andAUC, was approximately twofold smaller for both RG228 and RG242 relativeto rHCNTF, suggesting that these variants are distributed less widely.The limited number of animals used in these evaluations did not allowthe quantitative distinction of these values. However, these resultsclearly indicate that the distribution and disposition kinetics of RG228and RG242 after i.v. administration are not substantially different fromthose of rHCNTF.

[0169] After s.c. administration, RG228 and RG242 had a 2-3 fold longerabsorption phase (Ka) relative to rHCNTF (FIG. 10 and Table 2). Thedisposition phase of RG242 was also somewhat longer. The longer apparentterminal disposition phase of RG242 after s.c. dosing compared to i.v.administration may be attributed to the incomplete characterization ofthe terminal phase after the i.v. injection. Overall, the absolutebioavailability of RG228 (13.7%) and RG242 (10.9%) were comparable tothat of rHCNTF (6.0%), in view of the fact that in two previousindependent studies, the absolute bioavailability of rHCNTF was found tobe 14.2% (n=18) and 7.5% (n=8) (D. B. Lakings, et al., DSER 93/DMAP/006,“Dose Proportionality and Absolute Bioavailability of rHCNTF in the RatFollowing Subcutaneous Administration at Eight Dose Levels” (PhoenixInternational Project No. 920847) Nov. 10, 1993; D. B. Lakings, et al.,Dose Proportionality and Absolute Bioavailability of rHCNTF AdministeredSubcutaneously to Rats. AAPS Ninth Annual Meeting, San Diego, Calif.,November, 1994). Therefore, the bioavailabilities of rHCNTF, RG228 andRG242 are not significantly different within experimental error.

[0170] Efficacy and Toxicity In vivo—In control experiments, denervationof the soleus muscle resulted in a loss of 40% of muscle wet weight by 7days. This value is very accurate and reproducible, since it varies byonly 3% among independent experiments. Daily administration of rHCNTFresulted in a dose-dependent rescue of muscle wet weight at an ED₅₀=0.12mg/kg and a maximal effect at 0.3 mg/kg (FIG. 11). At the same time,even though animals continued to gain weight during the course of theseexperiments, they clearly did not gain as much as their vehicle-treatedcounterparts (p<0.01; FIG. 12), especially at the maximally efficaciousdoses.

[0171] In the course of several experiments conducted in parallel withrHCNTF, it was determined that the 63QR substitution resulted in a2-fold increase in potency in vivo (FIG. 11) but, also, a concomitant 2fold increase in toxicity (FIG. 12) In contrast, RG297, which carriesthe additional C17A and ΔC13 modifications, shows a 2.6 fold greaterpotency but the same toxicity relative to rHCNTF. Finally, RG242produced a 2.8 fold increased potency and an 2.4 fold decreased toxicityrelative to rHCNTF. These results are summarized in Table 3.

[0172] The relative therapeutic index (T.I.) for each of these compoundswas calculated as the ratio of the TD₂₅ and ED₅₀ values, normalized tothat of rHCNTF. While the T.I. of RG228 is equal to that of rHCNTF, theT.I. of RG297 and RG242 is 2.5 and 6.8 fold superior to that of rHCNTF,respectively.

[0173] Therefore, RG297 and RG242 have superior pharmacologicalproperties than rHCNTF. This is of great relevance to the clinicalsituation where decreased body weight is observed upon rHCNTF treatmentin humans.

[0174] One skilled in the art will recognize that other alterations inthe amino acid sequence of CNTF can result in a biologically activemolecule which may have enhanced properties. For example, applicant hasprepared a 17CS mutant which has a serine residue in place of thecysteine residue at position 17 and is biologically active. Applicanthas also prepared a biologically active quadruple mutant,17CA,ΔC13,63QR,64WA. Further CNTF mutants, all of which retainbiological activity, are set forth in Table 4. TABLE 1 AveragePharmacokinetic Parameters for rHCNTF, RG228 and RG242 after IntravenousAdministration to Rats at 100 μg/kg. Pharmacokinetic Compound ParameterrHCNTF RG242 RG228* n 1 2 1 C₀ (ng/ml) 726 2,175 NC AUC_(0-∞) (ng ·min/ml) 20,230 22,890 55,800 α (min⁻¹) 0.0492 0.0856 0.041 t_(1/2α)(min) 14 8 17 β (min⁻¹) 0.0106 0.0200 0.0176 t_(1/2β) (min) 65 35 39V_(area) (ml/kg) 470 220 204 CI_(T) (ml/min/kg) 4.9 4.4 3.6

[0175] TABLE 2 Average Pharmacokinetic Parameters for rHCNTF, RG228 andRG242 After Subcutaneous Administration to Rats at 200 μg/kgPharmacokinetic Compound Parameter rHCNTF RG242 RG228 n 2 2 1 C_(max)(ng/ml) 18 32 50 T_(max) (min) 30-45 30-45 60 AUC_(0-∞) (ng · min/ml)2,425 4,980 7,620 Absolute 6.0 10.9 13.7 Bioavailability k_(e) (min⁻¹)0.0133 0.0083 NC t_(1/2ke) (min) 52 82 NC k_(a) (min⁻¹) 0.0401 0.01800.0102 t_(1/2ka) (min) 17 39 68

[0176] TABLE 3 Efficacy, Toxicity and Therapeutic Index of rHCNTF andDerivatives ED₅₀ TD₂₅ Therapeutic Index Relative Therapeutic Compound(mg/kg) (mg/kg) (TD₂₅/ED₅₀) Index rHCNTF 0.12 0.087 0.72 1.0 RG228 0.0650.047 0.72 1.0 RG297 0.045 0.080 1.78 2.5 RG242 0.043 0.21 4.88 6.8

[0177] TABLE 4 Biological activity of rHCNTF variants on E8 chickciliary neurons. Potency units (1/EC₅₀) are shown relative to human CNTFwhich is assigned a value of 100. One potency unit is defined as thereciprocal ligand concentration showing the same biological activity as1 ng/ml rHCNTF. CNTF POTENCY rat 500.0 human 100.0 17CS 100.0 63QA 87.063QN 100.0 63QH 2.5 63QE <1 63QK 1.1 63QR 400.0 64WA 2.0 63QR64WA 9.063QR64WF 250.0 63QR64WH 25.0 63QR64WQ 10.0

Example 7 Efficacy of CNTF and Variants in Animal Models of Huntington'sDisease

[0178] Background

[0179] Glutamate receptor-mediated excitotoxicity has been hypothesizedto play a role in numerous neurodegenerative diseases, includingHuntington disease and motor neuron disease (DiFiglia, M. 1990, TrendsNeurosci. 13:286-289; Rothstein, et al., 1995, J. Neurochem.65:643-651). The predominant neuropathological feature of Huntingtondisease is a massive degeneration of the medium-sized, GABAergic,striatal output neurons, without substantial loss of striatalinterneurons (Albin, et al., 1989, Trends Neurosci. 12:366-375;Harrington, et al., 1991, J. Neuropathol. Exp. Neurol. 50:309). Thepreferential loss of striatal output neurons observed in Huntingtondisease, and the resulting dyskinesia, are mimicked in rodent or primatemodels in which an NMDA glutamate receptor agonist, quinolinic acid, isinjected into the striatum (DiFiglia, M., 1990, Trends Neurosci.13:286-289).

[0180] In the absence of a genetic animal model for HD, neuroscientistscontinue to rely on acute lesion models for investigation of the HDphenotype. The classic animal model of HD involves production of anexcitotoxic lesion of the rat striatum using a glutamate agonist of theNMDA-receptor class. In such lesion paradigms, injection of theneurotoxin directly into the striatum results in loss of the mediumsized intrinsic striatal neurons which utilize gamma-aminobutyric acid(GABA) as their neurotransmitter, with relative preservation of the twoclasses of striatal interneurons which utilize either acetylcholine orsomatostatin and neuropeptide Y as their neurotransmitters. Most recentstudies have relied upon intrastriatal injection of quinolinic acid,which seems to most faithfully reproduce the appearance of the HDstriatum.

[0181] Figueredo-Cardenas et al. (1994, Exp. Neurol 129:37-56) injectedquinolinic acid (QA), into the striatum in adult rats and 2-4 monthspost lesion explored the relative patterns of survival for the variousdifferent types of striatal projection neurons and interneurons as wellas the striatal efferent fibers in the different striatal projectionareas. The perikarya of all projection neuron types (striatopallidal,striatonigral, and striato-entopeduncular) were more vulnerable than thecholinergic interneurons. Among projection neuron perikarya, there wasevidence of differential vulnerability, with striatonigral neuronsappearing to be the most vulnerable. Examination of immunolabeledstriatal fibers in the striatal target areas indicated thatstriato-entopeduncular fibers better survived intrastriatal QA than didstriatopallidal or striatonigral fibers. The apparent order ofvulnerability observed in this study among projection neurons and/ortheir efferent fiber plexuses and the invulnerability observed in thisstudy of cholinergic interneurons is similar to that observed in HD.

[0182] In another animal model, systemic administration of3-nitropropionic acid (3-NP) leads to neuropathological changes similarto those seen in Huntington's disease (HD). Although the behavioralhypoactivity seen in these animals differs from the observedhyperactivity in most excitotoxic models of HD, 3-NP is considered bysome to provide a better model of juvenile onset and advanced HD. Theneuropathological effects of 3-NP include loss of intrinsic striatalcholinergic neurons, but some sparing of large AChE positive neurons,minimal damage of NADPH-diaphorase-containing neurons, and glialinfiltration (Borlongan et al., 1995, Brain Res. Bull. 365:49-56). Therehave been relatively few studies with 3-NP as a neurotoxic model of HD.Its faithfulness and utility remain to be explored.

[0183] Recent studies have begun to explore the relationship betweenexcitotoxic injury and the role of Huntingtin in the striatum. Striatalinjection of quinolinic acid in mice induces increased immunoreactivityfor Huntingtin in some remaining neurons but not in glial cells. Thisincrease is apparent in both neuronal cell bodies and in cell processesin the white matter six hours after excitotoxic challenge. ThusHuntingtin may be involved in the response to excitotoxic stress inthese neurons Tatter, et al., 1995, Neuroreport 6:1125-1129). Followingan initial increase between 1 h and 6 h, IT15 mRNA levels declined in apattern homologous to a group of neuron-specific genes. Decreased mRNAlevels after 24 h demonstrated that glial transcription is not activatedby neurodegeneration or gliosis. The 1 h and 24 h mRNA levels stronglysuggest that IT15 transcription preferentially localizes to degeneratingneurons. Carlock et al., 1995, Neuroreport 6:1121-1124.

[0184] Excitotoxic injury to the striatum also mimics certain of theaspects of cell death seen in HD brain (Beal et al., 1986, Nature321:168-171). In the neostriatum of individuals with HD, patterns ofdistribution of TUNEL-positive neurons and glia were reminiscent ofthose seen in apoptotic cell death during normal development of thenervous system; in the same areas, nonrandom DNA fragmentation wasdetected occasionally. Following excitotoxic injury of the rat striatum,internucleosomal DNA fragmentation (evidence of apoptosis) was seen atearly time intervals and random DNA fragmentation (evidence of necrosis)at later time points. In addition, EM detected necrotic profiles ofmedium spiny neurons in the lesioned rats. Thus, apoptosis occurs inboth HD and excitotoxic animal models. Furthermore, apoptotic andnecrotic mechanisms of neuronal death may occur simultaneously withinindividual dying cells in the excitotoxically injured brain. (Portera etal., 1995, J. Neuroscience 15:3775-3787).

[0185] The Tdt-mediated dUTP-biotin nick end labeling (TUNEL) techniquehas been investigated in preliminary studies of a variety of pathologicconditions of the human brain (e.g., gliomas, traumatic brain injury,Parkinson's disease, Parkinson's-Alzheimer's complex, multisystematrophy, striatonigral degeneration). Only Huntington's disease revealedsignificant and consistent labeling with this method. Thomas et al.,1995, Experimental Neurology 133:265-272). c-fos expression increasessoon after quinolinic acid injection, is widespread in rat brain, but iseffectively absent by 24 h postinjection. DNA fragmentation, however, islimited to striatum and is maximal at 24 h after injection. Theseresults demonstrate the sensitivity of in situ nick translation for thedetection of regional neuropathology and illustrate the temporal andspatial relationship of c-fos expression to excitotoxic neuronal death(Dure et al., 1995, Exp. Neurol. 133:207-214).

[0186] Excitotoxic lesions have also been used to explore possibletherapeutic avenues in HD. Excitotoxic striatal lesions induced byquinolinic acid, a model for Huntington's disease, have been used totest for neuroprotective actions of nerve growth factor (NGF) onstriatal cholinergic and GABAergic neurons in adult rats followingquinolinic acid lesion (150 mmol). Daily intrastriatal NGFadministration for one week increased the cellular expression of cholineacetyltransferase messenger RNA three times above control levels andrestored the levels of Trk A messenger RNA expression to control levels.In contrast to the protective effects on cholinergic cells, NGFtreatment failed to attenuate the quinolinic acid-induced decrease inglutamate decarboxylase messenger RNA levels. Thus, striatal glutamatedecarboxylase messenger RNA-expressing GABAergic neurons whichdegenerate in Huntington's disease are not responsive to NGF Frim, etal. (1993, J. Neurosurg. 78:267-273) implanted fibroblasts secreting NGFinto quinolinic-acid lesioned rat striata. They found thatpreimplantation of NGF-secreting fibroblasts placed within the corpuscallosum reduced the maximum cross-sectional area of a subsequentexcitotoxic lesion in the ipsilateral striatum by 80% when compared tothe effects of a non-NGF-secreting fibroblast graft, and by 83% whencompared to excitotoxic lesions in ungrafted animals (p<0.003).

[0187] Materials and Methods

[0188] Trophic Factors. Recombinant human BDNF, nerve growth factor(NGF) and NT-3, and recombinant rat CNTF were prepared in E. coli andcharacterized as described (Maisonpierre, et al., 1990, Science247:1446-1451; Masiakowski, et al., 1991, J. Neurochem. 57:1003-1012).Axokine1 (Ax1) is the designation for recombinant human CNTF with thefollowing modifications: substitutions of alanine for cysteine atposition 17 and arginine for glutamine at position 63, and deletion ofthe 13 C-terminal amino acids. This CNTF analog has enhanced solubility,is stable for at least a week at 37° C. in physiological buffer, andexhibits 4-5-fold greater potency in vitro relative to native human CNTF(Panayotatos et al., 1993, J. Biol. Chem. 268:19000-19003). AnimalTreatments. All animal procedures were conducted in strict compliancewith protocols approved by the institutional animal care and usecommittee.

[0189] Trophic factor delivery by osmotic pump. A 30-gauge osmotic pumpinfusion cannula and a 22-gauge guide cannula (5.0 and 2.2 mm long,respectively) were chronically implanted side-by-side into the lefthemisphere (stereotaxic coordinates AP 0.7, ML 3.2 relative to bregma;incisor bar 3.3 mm below the interaural line) in 250-300 g male,Sprague-Dawley rats under deep chloral hydrate (170 mg/kg) andpentobarbital (35 mg/kg) anesthesia. Thirty days later, the rats wereagain anesthetized and an Alzet osmotic minipump 2002 (two-week capacityat a delivery rate of 0.5 μl/hr), containing 0.1 M phosphate bufferedsaline (PBS) (pH 7.4), or PBS solutions of recombinant human NGF (0.9mg/ml), human BDNF (1 mg/ml), human NT-3 (1 mg/ml), rat CNTF (0.78mg/ml), or Ax1 (0.4 mg/ml) was connected by plastic tubing to theinfusion cannula and implanted subcutaneously (Anderson, et al., 1995,J. Comp. Neurol. 357:296-317). Due to the dead volume of the infusioncannula and tubing, the delivery of neurotrophic factor into the brainbegan about 1 day after pump implantation. Neurotrophins maintained inosmotic pumps at 37° C. for 12 days were completely stable, asdetermined by bioassay, and effective intrastriatal delivery of theneurotrophins was verified by immunohistochemical staining of sectionsfor the appropriate factor (Anderson, et al., 1995, J. Comp. Neurol.357:296-317). Three or four days after pump implantation, anesthetizedrats received an injection of quinolinic acid (50 mmol in 1 μl phosphatebuffer, pH 7.2, over 10 minutes) through the guide cannula using a 10-μlHamilton syringe with a 28-gauge blunt-tipped needle.

[0190] Trophic factor delivery by daily injection. A 22-gauge guidecannula (2.2 mm long) was chronically implanted into the left hemisphere(stereotaxic coordinates AP 0.5, ML 3.0) of anesthetized rats, asdescribed above. Beginning 1 week later, anesthetized rats received adaily intrastriatal injection of Ax1 (0.4 μg in 1 μl, over 10 minutes)or vehicle through the guide cannula using a Hamilton syringe. Ax1 wasinjected for 3 consecutive days before and 1 day after injection ofquinolinic acid, which was injected as described above. HistologicalProcedures and Analysis. Brains perfusion-fixed in 4% paraformaldehydewere collected 8 or 9 days after the quinolinic acid injection, and cutin the coronal plane into forty-micron thick sections that were stainedwith thionin. In each experiment, a series of 1 in 12 Nissl-stainedsections was evaluated by an investigator unaware of treatmentconditions, and the relative loss of medium-sized striatal neurons wasrated on the following scale: 0 (no neuron loss), 1 (clear but slightneuron loss), 2 (moderate neuron loss), 3 (severe but not total neuronloss), 4 (total loss of medium-sized neurons within the field of thequinolinic acid injection). In cases where neuron loss appearedintermediate to two criteria, a half score between the two closestscores was assigned. Neuron loss scores that were assigned independentlyby two different observers in the experiments using BDNF and NT-3 werewithin 0-0.5 points of each other for 40 of 42 rats (correlationcoefficient=0.8; p=0.0001).

[0191] In the experiment using CNTF, neuron loss also was evaluated bycounting neurons in sections taken 0.5 mm rostral to the infusioncannula. For each section, neurons were counted that intersected everyvertical line of a 10×10 sampling grid placed over seven fields, 0.4×0.4mm, within the treated striatum. The first field was located slightlylateral to the center of the striatum, at the center of a typicalquinolinic acid-induced lesion (i.e. immediately rostral to the tip ofthe infusion cannula). The six other fields were selected by movingdiagonally from the first field, twice each in the dorsomedial and theventromedial directions, and once each in the dorsolateral and theventrolateral directions. To control for possible variation in sectionthickness, seven fields in equivalent locations were sampled in thecontralateral striatum (approximately 600 neurons counted per 7 fields),and neuron survival was expressed as a percentage of neurons on thetreated side relative to the intact side. The results of actual neuroncounts (31 and 61% neuron loss for CNTF- and PBS-treated groups,respectively) showed close agreement with the results of the neuron lossscoring system (mean neuron loss scores of 1.67 and 3.25, respectively),as assessed by regression analysis (Spearman rank correlationcoefficient=0.82, p<0.05).

[0192] Differences between experimental groups and their respectivecontrol groups were evaluated by unpaired t-test.

[0193] Results

[0194] In a series of experiments, quinolinic acid (50 mmol) wasinjected into the left striatum of adult rats 3 or 4 days after thestart of intrastriatal infusion of neurotrophic factor by osmotic pump(nominal delivery rates: human NGF, 10.8 μg/day; human BDNF or NT-3,12.0 μg/day; rat CNTF, 9.4 μg/day). This dose of quinolinic acid istoxic to medium-sized striatal output neurons, which constitute over 90%of all striatal neurons, yet leaves the striatal populations ofcholinergic interneurons and parvalbumin/GABAergic interneurons largelyintact (Qin, et al., 1992, Experimental Neurology 115:200-211;Figueredo-Cardenas, et al., 1994, Exp. Neurol. 129:37-56). Microscopeanalysis of Nissl-stained sections from brains collected 8-9 days afterinjection of quinolinic acid demonstrated no significant sparing ofmedium-sized striatal neurons in BDNF-, NGF-, or NT-3-treated brains(FIG. 13). In an additional set of experiments, no neuron sparing wasapparent when quinolinic acid was injected 7 days after the start ofBDNF or NGF infusion.

[0195] In striking contrast, neuron survival was significantly greaterin rats treated with CNTF compared to rats treated with vehicle alone(FIG. 14), as determined by neuron counts that demonstrated a meanpercent survival (±SEM) of 69±17 and 29±11%, respectively (unpairedt-test, t(5)=2.12, p=0.04), or as assessed by assignment ofsemi-quantitative neuron loss scores (FIG. 15). Surviving neurons inCNTF-treated brains were disseminated throughout the striatal areaaffected by the quinolinic acid injection.

[0196] Given the favorable effect demonstrated by CNTF, a similarexperiment was conducted using a polypeptide CNTF receptor agonist,Axokine 1 (Ax1) (24). As observed after administration of CNTF, infusionof Ax1 (4.8 μg/day) resulted in significant sparing of medium-sizedstriatal neurons exposed to quinolinic acid (FIG. 15). This resultsupports the conclusion that CNTF receptor-mediated mechanisms effectprotection of striatal neurons from NMDA receptor-mediatedexcitotoxicity.

[0197] The neuroprotective effect of CNTF or Ax1 was achieved withoutapparent adverse effects on behavior or health, as indicated, forexample, by body weight. Body weights measured at the end of theexperiments were not significantly affected by CNTF or Ax1 treatment(unpaired t-test). The mean body weights (±SEM) of the trophicfactor-treated and the vehicle-treated groups in the CNTF experimentwere 369±20 g and 331±15 g, respectively, (p=0.21); mean body weights inthe Ax1 experiment were 431±26 g and 453±14 g, respectively, (p=0.44).

[0198] Two additional experiments were performed to determine whetherthe neuroprotective effect of CNTF receptor ligands might persist aftertermination of neurotrophic factor administration, and whether treatmentis effective when a lower dose of trophic factor is deliveredintermittently. In the first experiment, rats were infusedintrastriatally with Ax1 (4.8 μg/day) or vehicle for 3 days and thendelivery was terminated by removal of the osmotic pump. Quinolinic acidwas injected into the striatum 3 days thereafter (FIG. 16A). In thesecond experiment, rats received a daily intrastriatal injection of Ax1(0.4 μg/day) or vehicle for 3 days before and 1 day after intrastriatalinjection of quinolinic acid (FIG. 16B); thus these rats received atotal of only 1.6 μg Ax1. In both experiments, microscope analysis ofNissl-stained sections demonstrated significant sparing of medium-sizedstriatal neurons in Ax1-treated brains that was comparable to sparingseen when CNTF or Ax1 were infused continuously for the duration of theexperiment (FIG. 16).

[0199] Discussion

[0200] Since over 90% of the neurons in the striatum are medium-sized,GABAergic, striatonigral and striatopallidal projection neurons(Graybiel, A. M., 1990, TINS 13:244-254), the present results show thattreatment with CNTF or a CNTF receptor agonist protects striatal outputneurons against excitotoxic insult. Thus, CNTF is one of the firstpurified trophic factors demonstrated to protect striatal output neuronsafter pharmacological application in an adult animal model of Huntingtondisease. Among other factors characterized, only treatment with basicfibroblast growth factor has been reported to diminish the size of astriatal lesion induced by injection of N-methyl-D-aspartate (NMDA) ormalonic acid in adult and neonatal rats (Nozaki, et al., 1993, J. Cereb.Blood Flow Metab. 13:221-228; Kirschner, et al., 1995, J. Cereb. BloodFlow Metab. 15:619-623). Although NGF-secreting fibroblasts implantednear the striatum have been shown to protect medium-sized striatalneurons from quinolinic acid in rats (Frim, et al., 1993, NeuroReport4:367-370; Emerich, et al., 1994, Exp. Neurol. 130:141-150), we obtainedno survival-promoting effect on these neurons with purified NGF, inagreement with several earlier studies (Davies, et al., 1992, Neurosci.Lett. 140:161-164; Venero, et al., 1994, Neuroscience 61:257-268;Kordower, et al., 1994, Proc. Natl. Acad. Sci. USA 91:9077-9080). Thisfinding suggests that NGF is not the sole mediator of theneuroprotection provided by NGF-secreting fibroblasts. We did, however,observe that the large, darkly staining, presumably cholinergicinterneurons were more prominent in NGF-treated brains, as previouslyreported (Davies, et al., 1992, Neurosci. Lett. 140:161-164; Kordower,et al., 1994, Proc. Natl. Acad. Sci. USA 91:9077-9080; Perez-Navarro, etal., 1994, Eur. J. Neurosci. 6:706-711). Striatal expression of thehigh-affinity NGF receptor, TrkA, is restricted to cholinergicinterneurons (Steininger, et al., 1993, Brain Res. 612:330-335),consistent with the finding of a selective action of NGF on theseneurons, whereas the high-affinity receptors for BDNF and NT-3 (TrkB andTrkC) are expressed by numerous medium-sized striatal neurons (Altar, etal., 1994, Eur. J. Neurosci. 6:1389-1405). BDNF and NT-3 (unlike NGF)promote the survival and phenotypic differentiation of embryonic,GABAergic, striatal output neurons in vitro (Mizuno, et al., 1994 Dev.Biol. 165:243-256; Ventimiglia, et al., 1995, Eur. J. Neurosci).Moreover, these neurotrophins can protect certain neuron populationsfrom glutamate toxicity in vitro (Lindholm, et al., 1993, Eur. J.Neurosci. 5:1455-1464; Shimohama, et al., 1993, Neurosci. Lett.164:55-58; Cheng, et al., 1994, Brain Res. 640:56-67). Nevertheless,infusion of BDNF or NT-3 does not appear to protect striatal outputneurons against NMDA receptor-mediated excitotoxicity in vivo, althoughintracerebral infusion of BDNF or NT-3 at comparable doses elicitspronounced biological effects in the striatum and elsewhere in the brain(Lindsay, et al., 1994, TINS 17:182-190). The contrasting resultsbetween in vivo and in vitro studies may be explained by differences inneuron type (striatal vs. hippocampal, cortical or cerebellar), adifference in the developmental stage of the neurons (adult vs.embryonic), or the presence of glutamatergic synaptic input in vivo.

[0201] The neuroprotective effect displayed by CNTF receptor ligands mayoccur through direct action on medium-sized striatal neurons, sincethere is abundant expression of mRNA for components of the CNTF receptor(CNTFRα, LIFRβ, gp130) in the striatum (Ip, et al., 1993, Neuron10:89-102; Rudge, et al., 1994, Eur. J. Neurosci. 6:693-705). Potentialmechanisms might include alteration of the expression or function ofglutamate receptors, thereby modifying neuron sensitivity toglutamatergic stimulation, or enhancement of the neuron's capacity toregulate the cytosolic concentration of calcium ion, an increase inwhich is thought to be a critical event initiating the neurodegenerativeprocess (Choi, D. W., 1988, Neuron 1:623-634). The possibility that CNTFacts as a glutamate receptor antagonist to block quinolinic acidtoxicity is unlikely, since CNTF does not block the toxic effects ofglutamate in vitro (Mattson, et al., 1995, J. Neurochem. 65:1740-1751).On the other hand, CNTF receptor ligands could potentially actindirectly, via other components of the striatum. For example,elimination of nigral or cortical input to the striatum prior toexposure to quinolinic acid results in a significant reduction in theloss of striatal neurons (DiFiglia, M., 1990, Trends Neurosci.13:286-289; Buisson, et al., 1991, Neurosci. Lett. 131:257-259)indicating that the combined actions of exogenous toxin and endogenousneurotransmitters are required to induce cell death. Thus, a reductionin synaptic transmission at either glutamatergic or dopaminergicsynapses would likely protect striatal neurons from an injection ofquinolinic acid. Although astrocytes do not normally express detectableCNTFRα in vivo (Ip, et al., 1993, Neuron 10:89-102), astrocytes doexpress all CNTF receptor components when activated by brain injury orwhen maintained in vitro (Rudge, et al., 1994, Eur. J. Neurosci.6:693-705). Furthermore, intracerebral delivery of CNTF appears toactivate astrocytes 10-48 hours after exposure, as indicated byincreased content of glial fibrillary acidic protein and its mRNA(Levison, et al., 1995, Soc. Neurosci. Abst. 21:497; Winter, et al.,1995, Proc. Natl. Acad. Sci. USA 92:5865-5869). Whether activatedindirectly or directly by CNTF, astrocytes might promote neuron survivalthrough enhanced sequestration of excitatory amino acids or by releaseof substances that protect neurons.

[0202] The striatal neuron populations protected from excitotoxic damageby CNTF receptor-mediated events in the present study are the same typesselectively lost in Huntington disease (Albin, et al., 1989, TrendsNeurosci. 12: 366-375). A potential link between excitotoxic stimulationand increased expression of the Huntington disease gene has recentlybeen suggested (Carlock, et al., 1995, NeuroReport 6:1121-1124; Tatter,et al., 1995, NeuroReport 6:1125-1129). While extensive studies are inprogress to identify the mechanisms which lead to Huntington disease,existing lines of evidence clearly implicate a role for NMDAreceptor-mediated excitotoxicity (DiFiglia, M., 1990, Trends Neurosci.13:286-289).

Example 8 PEGylation of Axokine Protein

[0203] Pegylation of proteins has been shown to increase their in vivopotency by enhancing stability and bioavailability while minimizingimmunogenicity. It is known that the properties of certain proteins canbe modulated by attachment of polyethylene glycol (PEG) polymers, whichincreases the hydrodynamic volume of the protein and thereby slows itsclearance by kidney filtration. (See, Clark, R., et al., 1996, J. Biol.Chem. 271: 21969-21977). We have generated PEGylated Axokine bycovalently linking polyethylene glycol (PEG) to Ax-13. We have alsodeveloped a purification methodology to separate different PEGylatedforms of Axokine from unmodified molecules. PEGylated Ax-13 has bettersolubility and stability properties, at physiological pH, thanunPEGylated Ax-13. PEGylation has been shown to greatly enhancepharmacokinetic properties of Ax-13 and would be expected to similarlyenhance the properties of other Axokine molecules.

[0204] Purified Ax-13 derived from E. coli was used for these studies.20 kD mPEG-SPA was obtained from Shearwater Polymers, Bicine from Sigma,and Tris-Glycine precast gels from Novex, CA. A small scale reactionstudy was set up to determine reaction conditions. 20 kD mPEG SPA wasreacted with purified Ax-13 at a final concentration of 0.6 mg/ml, at 4°C. in an amine-free buffer at a pH of 8.1. Molar ratios of PEG toprotein were varied and two reaction times were used. The reaction wasstopped by the addition of a primary amine in large excess. Reactionproducts were analyzed by reducing SDS-PAGE. The predominant modifiedspecies ran at a molecular weight of approximately 60 kD. Higher ordermodified bands that ran at higher molecular weights were also seen.Based on this study, an overnight reaction at a PEG-to-protein ratio of4 was chosen.

[0205] Ax-13 at 0.6 mg/mL was reacted with 20 kD mPEG SPA in a Bicinebuffer overnight at 4° C. at a pH of 8.1. The reaction was stopped bythe addition of a primary amine in large excess. The reaction productwas diluted with a low salt buffer and applied to an ion-exchangecolumn. The column was washed with a low salt buffer and eluted with aNaCl gradient. A good separation between higher order forms (apparentMW>66 kD on SDS-PAGE), a distinct modified species that ran at about 60kD and unmodified Ax-13 was obtained. Fractions corresponding to the 60kD band were tested in a Bioassay. A very faint band of unmodified Ax-13was noticed in the fractions corresponding to the 60 kD band. To ensurethat the bioassay results were not influenced significantly by thismaterial, the 60 kD band was further purified by Size exclusionchromatography (SEC) that resulted in baseline separation betweenunmodified Ax-13 and the 60 kD band. The purified modified Ax-13 wastested in a Bioassay and the results were indistinguishable from thoseobtained with the material prior to SEC.

Example 9 Construction of Ax-15 Expression Plasmid pRG643

[0206] The expression plasmid pRG632 is a high copy plasmid that encodesampicillin resistance and the gene for human CNTF-C17A,Q63R,ΔC13 (alsoreferred to herein as either Ax1 or Ax-13) with a unique Eag Irestriction enzyme recognition sequence 3′ to the stop codon. Thisplasmid was used to construct a human CNTF mutation C17A,Q63R,ΔC15(designated Ax-15) by PCR amplification of a 187 bp BseR I-Eag1 DNAfragment that incorporates the ΔC15 mutation. The 5′ primer {ΔC15-5′(5′-CCAGATAGAGGAGTTAATGATACTCCT-3′)} encodes the BseR I site and the 3′primer ΔC15-3′ {(5′-GCGTCGGCCGCGGACCACGCTCATTACCCAGTCTGTGAGAAGAAATG-3′)} encodes the C-terminus of the Ax-15 gene ending at Gly185followed by two stop codons and an Eag I restriction enzyme recognitionsequence.

[0207] This DNA fragment was digested with BseR I and Eag I and ligatedinto the same sites in pRG632. The resulting plasmid, pRG639, encodesthe gene for Ax-15 (human CNTF C17A,Q63R,ΔC15). The ΔC15 mutation wasthen transferred as a 339 bp Hind III-Eag I DNA fragment into thecorresponding sites within pRG421, a high copy number expression plasmidencoding the gene for kanamycin resistance and human CNTFC17A,Q63R,ΔC13. The resulting plasmid, pRG643, encodes the gene forAx-15 under transcriptional control of the lacUV5 promoter, and conferskanamycin resistance. The Ax-15 gene DNA sequence was confirmed bysequence analysis.

Example 10 Small Scale Expression and Purification of Ax-15 Protein

[0208]E. coli strain RFJ141 containing pRG639 was grown in LB medium andexpression of Ax-15 protein was induced by the addition of lactose to 1%(w/v). Induced cells were harvested by centrifugation, resuspended in 20mM Tris-HCl, pH 8.3, 5 mM EDTA, 1 mM DTT, and lysed by passage through aFrench pressure cell at 10,000 psi. The cell lysate was centrifuged andthe pellet was resuspended in 8 M guanidinium-HCl, 50 mM Tris-HCl, pH8.3, 0.05 mM EDTA then diluted with 5 volumes of 50 mM Tris-HCl, pH 8.3,0.05 mM EDTA (Buffer A) followed by dialysis against Buffer A. Thedialysate was loaded onto a Q-sepharose column equilibrated with BufferA. The Ax-15 protein was eluted by a linear gradient to 1 M NaCl in 10column volumes of buffer. Fractions containing Ax-15 were pooled andbrought to 1 M (NH₄)₂SO₄ by the slow addition of solid (NH4)₂SO₄ whilemaintaining the pH at 8.3 by the addition of NaOH. The pool was loadedonto a phenyl-sepharose column equilibrated with 1 M (NH₄)₂SO₄ in BufferA. The column was washed with 0.5 M (NH₄)₂SO₄ in Buffer A, and the Ax-15protein was eluted by a linear gradient of decreasing (NH₄)₂SO₄concentration. Fractions containing Ax-15 protein were pooled, dialyzedagainst 5 mM NaPO₄, pH 8.3, then concentrated by ultrafiltration. Theconcentrated pool was fractionated on an Sephacryl S-100 columnequilibrated with 5 mM NaPO₄, pH 8.3.

Example 11 Large Scale Expression and Purification of Ax-15 Protein

[0209] A recombinant, kanamycin resistant E. Coli strain RFJ141expressing the Ax-15 protein under lac promoter control (pRG643) wasgrown to an intermediate density of 30-35 AU₅₅₀ (Absorbance@550 nM) in aminimal salts, glucose medium containing 20 μg/ml Kanamycin. Expressionof Ax-15 protein was induced by addition of IPTG (isopropylthiogalactoside) to 1.0 mM and the fermentation was continued for anadditional 8 hr. Ax-15 protein was expressed as insoluble inclusionbodies following IPTG induction. Post-induction, cells were harvested,cell paste concentrated, and buffer exchanged to 20 mM Tris, 1.0 mM DTT,5.0 mM EDTA, pH 8.5 via AGT 500,000 molecular weight cut off (mwco)hollow fiber diafiltration (ACG Technologies, Inc.). Inclusion bodieswere released from the harvested cells by disruption via repeatedpassage of cooled (0-10° C.) cell paste suspension through a continuousflow, high pressure (>8,000 psi) Niro Soavi homogenizer. The homogenatewas subjected to two passages through a cooled (4-8° C.) continuousflow, high speed (>17,000×G) Sharples centrifuge (source) to recoverinclusion bodies. Recovered inclusion bodies were extracted in 8.0 MGuanidine HCL with 1.0 mM DTT. The Ax-15 protein/guanidine solution wasdiluted into 50 mM Tris-HCl, 1.0 mM DTT, 0.05 mM EDTA, pH 8.0-8.3, anddiafiltered versus diluent buffer with AGT 5,000 mwco hollow fiberfilters (ACG Technologies, Inc.). The resulting solution, containingrefolded Ax-15, was filtered through a Microgon 0.22 μm hollow fiberfilter (ACG Technologies, Inc.) prior to chromatographic purification.

Example 12 Column Chromatographic Purification of Refolded Ax-15

[0210] The filtered Ax-15 solution described above was loaded onto a16.4 L DEAE Sepharose (Pharmacia) column at 10.9 mg/ml resin and washedwith 50 L of 50 mM Tris, pH 8.0-8.3, 1.0 mM DTT, and 0.05 mM EDTAbuffer. The Ax-15 protein was eluted from the column with a 120 mM NaClstep in the same Tris buffer. Eluate exceeding a previously established280 nM absorbance criteria of 40% maximum A₂₈₀ on the ascending portionof the peak and 20% of maximum A₂₈₀ on the descending portion of thepeak was pooled and either stored frozen (−30° C.) or used in the nextstep of the purification procedure. Pooled eluted Ax-15 protein wasadjusted to 1.0 M ammonium sulfate by gradual addition of the solidcompound, maintaining the pH at 8.0-8.3. The solution was filteredthrough a 0.22 μm Sartorious capsule filter, loaded onto a 12.5 L phenylSepharose HP (Pharmacia) column at 8.24 mg/ml of resin, and washed with55 L of 1.0 M ammonium sulfate in 50 mM Tris buffer with 0.05 mM EDTA,pH 8.0-8.3. Following a 12.0 L wash with 250 mM ammonium sulfate in thesame Tris buffer, the Ax-15 protein was eluted with a 125 mM ammoniumsulfate, Tris buffer wash step. Eluate exceeding previously established280 nM absorbance criteria of 100% maximum A₂₈₀ on the ascending portionof the peak and 20% of maximum A₂₈₀ on the descending portion of thepeak was pooled. Eluate was simultaneously diluted 1:4 into 50 mM Tris,pH 8.0-8.3 buffer without salt to reduce its conductivity. Pooledmaterial was stored frozen (−30° C.) or used in the following step.Pooled hydrophobic interaction chromatography (HIC) material wasconcentrated to 25 L and diafiltered versus 5.0 mM sodium phosphatebuffer pH 8.0-8.3 using a 5,000 mwco AGT hollow fiber filter (ACGTechnologies, Inc.). The pH was adjusted to 7.0-7.2 immediately prior tosulfyl propyl fast flow (SP FF) sepharose chromatography by gradualaddition of concentrated (85%) phosphoric acid. The pH-adjusted pooledmaterial was loaded onto a 7.7 L SP FF sepharose (Pharmacia) column to9.0 mg/ml of resin and washed with a minimum of 25 L of 5.0 mM sodiumphosphate buffer, pH 7.0. The Ax-15 protein was eluted with a 77.0 Lstep of 5.0 mM sodium phosphate, 130 mM NaCl, pH 7.0-7.2. The eluate wassimultaneously diluted 1:5 into 10.0 mM sodium phosphate, pH 9.0-9.2buffer without salt to reduce conductivity and increase pH. Peakmaterial exceeding 20% maximum A₂₈₀ on the ascending portion of the peakand 20% of the maximum A₂₈₀ on the descending portion of the peak waspooled. Pooled Ax-15 protein was stored frozen (−30° C.) or used in thefollowing step. Pooled SP FF sepharose Ax-15 protein was concentratedand diafiltered versus 5.0 mM sodium phosphate, pH 8.0-8.3 buffer with a5,000 mwco AGT hollow fiber filter (ACG Technologies, Inc.). The pool(24.66 g) was concentrated to ≦5.0 L. Concentrated, diafiltered Ax-15protein was loaded onto a 50 L S-100 Sephacryl (Pharmacia) sizing columnand eluted with 250 L of the same 5.0 mM sodium phosphate buffer, pH8.0-8.3. Peak material exceeding 40% maximum A₂₈₀ on the ascendingportion of the peak and 40% of the maximum A₂₈₀ on the descendingportion of the peak was pooled. The pooled Ax-15 protein was filteredthrough Millipak 0.22 μm filters and stored at −80° C. prior todispensing or formulation. The amino acid sequence of Ax-15 producedfollows. Alternatively, one could produce a sequence which contains aMethionine residue before the initial Alanine.        9         19         29         39         49         59   *   *     *    *     *    *     *    *     *    *     *    *AFTEHSPLT PHRRDLASRS IWLARKIRSD LTALTESYVK HQGLNKNINL DSADGMPVAS        69         79         89         99        109        119    *    *     *    *     *    *     *    *     *    *     *    *TDRWSELTEA ERLQENLQAY RTFHVLLARL LEDQQVHFTP TEGDFHQAIH TLLLQVAAFA       129        139        149        159        169        179    *    *     *    *     *    *     *    *     *    *     *    *YQIEELMILL EYKIPRNEAD GMPINVGDGG LFEKKLWGLK VLQELSQWTV RSIHDLRFIS     *SHQTG

[0211] Methionine⁺        10         20         30         40         50         60    *    *     *    *     *    *     *    *     *    *     *    *MAFTEHSPLT PHRRDLASRS IWLARKIRSD LTALTESYVK HQGLNKNINL DSADGMPVAS        70         80         90        100        110        120    *    *     *    *     *    *     *    *     *    *     *    *TDRWSELTEA ERLQENLQAY RTFHVLLARL LEDQQVHFTP TEGDFHQAIH TLLLQVAAFA       130        140        150        160        170        180    *    *     *    *     *    *     *    *     *    *     *    *YQIEELMILL EYKIPRNEAD GMPINVGDGG LFEKKLWGLK VLQELSQWTV RSIHDLRFIS     *SHQTG

Example 13 Use of Ax-15 to Treat Obesity

[0212] Animal Models:

[0213] Normal Mice

[0214] Normal (8 weeks) C57BL/6J mice were obtained from Taconic. Themice received daily subcutaneous injections of vehicle or Ax-15. Theanimals were weighed daily and food intake over 24-hours was determinedbetween days 3 and 4.

[0215] ob/ob Mice

[0216] As a result of a single gene mutation on chromosome 6, ob/ob miceproduce a truncated, non-functional gene product (Leptin). These miceare hyperphagic, hyperinsulinemic, and markedly obese.

[0217] C57BL/6J ob/ob mice were obtained from Jackson Laboratory andused for experiments at 12-14 weeks of age. The mice received dailysubcutaneous injection of vehicle, Ax-15, or leptin. Pair-fed group wasgiven the average amount (g) of food consumed by animals treated withAx-15 (0.3 mg/kg). Body weights were obtained daily and food intake over24-hours was determined between days 3 and 4. On day 8, the animals weresacrificed and carcass analysis was performed.

[0218] Diet-Induced Obesity (DIO) Mice

[0219] AKR/J mice have been shown to be very susceptable to diet inducedobesity by increasing body fat content. Although the gene-environment(diet) interaction is not completely known regarding this kind ofdietary obesity, like in human obesity, the genotype is polygenic.

[0220] AKR/J mice were obtained from Jackson Laboratory and put on ahigh fat diet (45% fat; Research Diets) at age 10-12 weeks old. Allexperiments commenced after 7 weeks on high fat diet. The mice receiveddaily subcutaneous injection of vehicle, Ax-15, or Leptin. Pair-fedgroup was given the average amount (g) of food consumed by animalstreated with Ax-15 (0.1 mg/kg). The animals were weighed daily and foodintake over 24-hours was determined between days 3 and 4. On day 8, theanimals were sacrificed and sera were obtained for insulin andcorticosterone measurements.

[0221] II. Reagents:

[0222] Recombinant human Ax-15 was manufactured as set forth above andLeptin was purchased from R & D Systems.

[0223] Results

[0224] Normal Mice

[0225] Ax-15 reduced body weight in normal mice in a dose dependentmanner. In 6 days, the animals lost approximately 4%, 11%, and 16% oftheir body weight at 0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg, respectively(FIG. 17).

[0226] ob/ob Mice

[0227] There was a dose related (0.1 mg/kg-3 mg/kg) decrease in bodyweight after Ax-15 treatment in ob/ob mice (FIG. 18). At a dose range of0.1 mg/kg to 3 mg/kg, there was a 8%-25% reduction of body weight.Animals pair-fed to a specific dose of Ax-15 (0.3 mg/kg) showedequivalent loss of body weight as the mice given that dose of Ax-15,suggesting food intake is the primary cause of weight reduction.

[0228] Leptin was also effective in decreasing body weight in ob/obmice. At 1 mg/kg, leptin decreased body weight 6% in 7 days, following acourse almost identical to that of Ax-15 given at 0.1 mg/kg (FIG. 18).

[0229] Carcass analysis showed that there was a significant reduction oftotal body fat with Ax-15 and Leptin treatments as well as in pair-fedcontrols (Table 5). There was a small but non-significant loss of leanmass in all groups as compared to vehicle control animals. Micereceiving only food restriction (pair-fed) had a fat/lean mass ratio nodifferent from vehicle controls, indicating that they lost fat and leanmass equally. However, the Ax-15 and Leptin treated animals showedpreferential loss of body fat as reflected by a decrease in fat/leanmass ratio (Table 5).

[0230] DIO Mice

[0231] Ax-15 reduced body weight in DIO mice dose dependently. Withinone week, the animals lost approximately 14%, 26%, and 33% of their bodyweight when given Ax-15 at 0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg,respectively (FIG. 19). Comparing the effects of the Ax-15 treatment andthe pair-fed control animals, there was a small but significantdifference between the 2 groups, suggesting that decrease food intakewas probably the primary, although not the only, cause of weight losswith Ax-15 treatment. Indeed, Ax-15 significantly attenuated the obesityassociated hyperinsulinemia in DIO mice, whereas merely reducing foodintake (pair-fed) did not (FIG. 20A). In addition, Ax-15 did not causeelevation of corticosterone levels, which is a common effect of foodrestriction (FIG. 20B).

[0232] It is of interest to note that when Ax-15 was administered in thesame dose range (0.1-1 mg/kg), DIO mice lost more than twice the bodyweight when compared to normal mice (see FIG. 17). This highersensitivity of diet-induced obese animals to Ax-15 suggests thatadiposity may regulate the efficacy of Ax-15 such that Ax-15 will notcause continuous weight loss after adiposity is normalized.

[0233] DIO mice are leptin resistant; no weight loss effect was observedin these animals with daily injection of leptin (1 mg/kg; FIG. 19).

[0234] We conclude as follows:

[0235] 1. Ax-15 caused weight loss in normal mice in a dose dependentmanner.

[0236] 2. Ax-15 induced weight loss in ob/ob mice in a dose dependentmanner. Ax-15 (0.1 mg/kg) was as effective as Leptin (1 mg/kg) incausing weight loss in ob/ob mice. Both Ax-15 and Leptin treatments, butnot pair-fed, preferentially reduced total body fat over lean mass.

[0237] 3. Ax-15 caused weight loss in diet-induced obesity mice in adose dependent manner, whereas Leptin was ineffective. Ax-15 treatmentattenuated obesity associated hyperinsulinemia in DIO mice; this effectwas not observed in pair-fed control animals. In addition, Ax-15 wasmore effective in inducing weight loss in DIO mice than normal or ob/obmice. Taken together, our results suggest a specific useful applicationof Ax-15 in the treatment of leptin resistant obesity, such as type 11diabetes associated obesity.

[0238] 4. The effectiveness of Ax-15 in reducing body weight in leptinresistant mouse model suggests that Ax-15 may also be effective inreducing body weight in obese humans who are resistant or unresponsiveto Leptin. TABLE 5 Results from carcass analysis of ob/ob mice Lean Fatg mass g Fat:Lean mass Mean 34.77 4.79 7.26 Vehicle sem 1.41 0.24Pair-fed to Ax-15 0.3 mg/kg 29.36 4.03 7.28 0.93 0.07 Ax-15 0.1 mg/kg30.22 4.38 6.9 0.59 0.13 Ax-15 0.3 mg/kg 26.77 4.03 6.64 0.66 0.08 Ax-151 mg/kg 23.29 3.35 6.95 0.87 0.12 Ax-15 3 mg/kg 23 3.5 6.57 0.53 0.12Leptin 1 mg/kg 28.89 4.73 6.11 0.89 0.1

[0239] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

We claim:
 1. Modified human ciliary neurotrophic factor having themodification Cys17→Ala, Gln63→Arg, and a deletion of the terminal 15amino acid residues.
 2. An isolated nucleic acid molecule encoding themodified human ciliary neurotrophic factor of claim
 1. 3. A recombinantDNA molecule of claim 2, operatively linked to an expression controlsequence.
 4. A host cell transformed with the recombinant DNA moleculeof claim
 3. 5. A method for producing a modified ciliary neurotrophicfactor molecule comprising: (a) growing a recombinant host cellcontaining the DNA molecule of claim 3, so that the DNA molecule isexpressed by the host cell to produce the modified ciliary neurotrophicfactor molecule and (b) isolating the expressed, modified ciliaryneurotrophic factor molecule.
 6. The method according to claim 5,wherein said host cell is a eukaryotic cell.
 7. The method according toclaim 5, wherein said host cell is a prokaryotic cell.
 8. A compositioncomprising the modified ciliary neurotrophic factor molecule of claim 1,and a carrier.
 9. A method of treating a disease or disorder of thenervous system comprising administering the modified ciliaryneurotrophic factor of claim
 1. 10. The method according to claim 9,wherein said disease or disorder is a degenerative disease.
 11. Themethod according to claim 9, wherein said disease or disorder involvesthe spinal cord, motor neurons, cholinergic neurons or cells of thehippocampus.
 12. The method according to claim 9, in which the diseaseor disorder of the nervous system comprises damage to the nervous systemcaused by an event selected from the group consisting of trauma,surgery, infarction, infection, malignancy and exposure to a toxicagent.
 13. The method according to claim 9, wherein said disease ordisorder involves muscle atrophy.
 14. A method of protecting striatalneurons from degeneration comprising treating said striatal neurons withan effective amount of the modified ciliary neurotrophic factor ofclaim
 1. 15. A method of treating Huntington's disease comprising directadministration to the central nervous system of the modified ciliaryneurotrophic factor of claim
 1. 16. A method of inducing weight loss ina mammal comprising administration of the modified ciliary neurotrophicfactor of claim
 1. 17. The method of claim 16, wherein the mammal is ahuman.
 18. The method claim 17, used in the treatment of morbid obesityor obesity of a genetically determined origin.
 19. A method ofpreventing the occurrence of gestational or adult onset diabetes in ahuman comprising administration of the modified ciliary neurotrophicfactor of claim
 1. 20. A method of treating gestational or adult onsetdiabetes in a human comprising administration of the modified ciliaryneurotrophic factor of claim
 1. 21. The method according to any one ofclaims 15 to 20, wherein said administration is via a route of deliveryselected from the group consisting of intravenous, subcutaneous,intramuscular, intrathecal, intracerebroventricular andintraparenchymal.
 22. The method according to any one of claims 15 to20, wherein said administration is via implantation of cells thatrelease the modified ciliary neurotrophic factor.